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42867
Human Reactive Cell Death and Autophagy Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Human Reactive Cell Death and Autophagy Antibody Sampler Kit #42867

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Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 1

Flow cytometric analysis of Jurkat cells, untreated (blue) or treated with etoposide #2200 (green), using Cleaved Caspase-3(Asp175) (5A1E) Rabbit mAb compared to a nonspecific negative control antibody (red).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 2

Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated (green), using Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 3

Western blot analysis of HT-29 cells, untreated (-) or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), human TNF-α (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-RIP (Ser166) (D1L3S) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 4

Confocal immunofluorescent analysis of HT-29 cells, untreated (left), pre-treated with Z-VAD (20 μM, 30 min) followed by treatment with SM-164 (100 nM) and Human Tumor Necrosis Factor-α (hTNF-α) #8902 (20 ng/mL, 6 hr; center), or pre-treated with Z-VAD followed by treatment with SM-164 and hTNF-α and post-processed with λ-phosphatase (right), using Phospho-RIPK3 (Ser227) (D6W2T) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 5

Western blot analysis of HT-29 cells, untreated (-), or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), human TNF-α (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb (upper), or MLKL (D2I6N) Rabbit mAb #14993 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 6

Immunohistochemical analysis of paraffin-embedded human ductal breast carcinoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 7

Confocal immunofluorescent analysis of THP-1 cells, differentiated with TPA #4174 (80 nM, 24 hr) and subsequently treated with (right) or without (left) Lipopolysaccharides (LPS) #14011 (1 μg/ml, 6 hr), using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 8

Confocal immunofluorescent analysis of MIA PaCa-2 (upper) and C2C12 (lower) cells, untreated (left), nutrient-starved with EBSS (2 hr; middle), or chloroquine treated (50 μM, 24 hr; right), using LC3B (E5Q2K) Mouse mAb (green) and β-Actin (D6A8) Rabbit mAb #8457 (red). Blue = Hoechst 33342 #4082 (fluorescent DNA dye).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 9

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with chloroquine (50 μM overnight; right), using SQSTM1/p62 (D5L7G) Mouse mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 10

Western blot analysis of extracts from HeLa cells (lane 1) or SQSTM1 knock-out cells (lane 2) using SQSTM1/p62 (D5L7G) Mouse mAb #88588 (upper), and β-actin (D6A8) Rabbit mAb #8457 (lower). The absence of signal in the SQSTM1 knock-out HeLa cells confirms specificity of the antibody for SQSTM1.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 11

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 12

Confocal immunofluorescent images of HT-29 cells, untreated (left) or Staurosporine #9953 treated (right) labeled with Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb (green). Actin filaments have been labeled with Alexa Fluor® 555 phalloidin #8953 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 13

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with Staurosporine #9953 (right), using Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb (green). Actin filament were labeled with DY-554 phalloidin. Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 14

Western blot analysis of HT-29 cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), and SM-164 (100 nM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb. To confirm phospho-specificity, membranes were either untreated (left) or treated with Calf Intestinal Phosphatase (CIP; right).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 15

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb in the presence of non-cleaved Gasdermin D peptide (left) or Asp275 cleavage-specific Gasdermin D peptide (right).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 16

Immunoprecipitation of Cleaved-IL-1β (Asp116) from extracts of THP-1 cells differentiated with TPA #4147 (80 nM, overnight) followed by treatment with Lipopolysaccharides (LPS) #14011 (1 μg/ml, 6 hr). Lane 1 is 10% input, lane 2 is precipitated with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is precipitated with Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb. Western blot was performed using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 17

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 18

Immunohistochemical analysis of paraffin-embedded human colon carcinoma using SQSTM1/p62 (D5L7G) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 19

Immunohistochemical analysis of paraffin-embedded mouse embryo, using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb in the presence of control peptide (left) or Cleaved Caspase-3 (Asp175) Blocking Peptide (#1050) (right).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 20

Immunohistochemical analysis of paraffin-embedded human tonsil using Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 21

Western blot analysis of HT-29 cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), SM-164 (100 nM, 7 hr; +), and necrostatin-1 (Nec-1, 50 μM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb (upper), RIP3 (E1Z1D) Rabbit mAb #13526 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 22

Immunohistochemical analysis of paraffin-embedded human squamous cell lung carcinoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 23

Western blot analysis of recombinant Human Interleukin-1β (hIL-1β) #8900 using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 24

Immunohistochemical analysis of paraffin-embedded human esophageal carcinoma using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 25

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using SQSTM1/p62 (D5L7G) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 26

Immunohistochemical analysis using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb on SignalSlide® Cleaved Caspase-3 IHC Controls #8104 (paraffin-embedded Jurkat cells, untreated (left) or etoposide-treated (right)).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 27

Western blot analysis of extracts from HeLa cells, untreated or treated with Staurosporine #9953 (1 μM, 3 hr), Jurkat cells, untreated or etoposide-treated (25 μM, overnight), and THP-1 cells, untreated or cycloheximide-treated (CHX, 10 μg/ml, overnight) followed by treatment with TNF-α #8902 (20 ng/ml, 4 hr), using Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb (upper), or total PARP Antibody #9542 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 28

Western blot analysis of HT-29 cells or HT-29 RIPK1 KO cells, untreated (-) or treated with a combination of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Human Tumor Necrosis Factor-α #8902 (hTNF-α, 20 ng/ml, 7 hr; +), and SM-164 (100 nM, 7 hr; +), using Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb (upper), RIP3 (E1Z1D) Rabbit mAb #13526 (middle) or β-Actin (D6A8) Rabbit mAb #8457 (lower). HT-29 RIPK1 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston, MA.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 29

Immunohistochemical analysis of paraffin-embedded human non-Hodgkin's Lymphoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 30

Western blot analysis of extracts from cells or media collected from THP-1 cells, differentiated with TPA #4147 (80 nM, overnight) and subsequently treated with (+) or without (-) Lipopolysaccharides (LPS) #14011 (1 μg/ml, 6 hr), using Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 31

Immunohistochemical analysis of paraffin-embedded human lung carcinoma using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 32

Immunohistochemical analysis of paraffin-embedded human endometrioid adenocarcinoma using SQSTM1/p62 (D5L7G) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 33

Immunohistochemical staining of paraffin-embedded mouse embryo, showing cytoplasmic localization in apoptotic cells, using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 34

Immunohistochemical analysis of paraffin-embedded human prostate carcinoma using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 35

Immunohistochemical analysis of paraffin-embedded human non-Hodgkin's lymphoma using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 36

Immunohistochemical analysis of paraffin-embedded HDLM-2 (left) and Daudi (right) cell pellets using SQSTM1/p62 (D5L7G) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 37

Immunoprecipitation of extracts from Jurkat cells, untreated or etoposide-treated (25uM, 5hrs), using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb. Western blot was performed using the same antibody.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 38

Immunohistochemical analysis of paraffin-embedded human spleen (left, positive) and skeletal muscle (right, negative) using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 39

Immunohistochemical analysis of paraffin-embedded normal human brain using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 40

Immunohistochemical analysis of paraffin-embedded human inflamed gall bladder using SQSTM1/p62 (D5L7G) Mouse mAb in the presence of control peptide (left) or antigen-specific peptide (right).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 41

Western blot analysis of extracts from C6 (rat), NIH/3T3 (mouse), and Jurkat (human) cells, untreated or treated with staurosporine #9953 (1uM, 3hrs) or etoposide #2200 (25uM, 5hrs) as indicated, using Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 42

Immunoprecipitation of Cleaved Gasdermin D (Asp725) from THP-1 cells differentiated with TPA #4174 (50 ng/ml, overnight) and then treated with LPS #14011 (5 μg/ml, 6 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb. Western blot was performed using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 43

Immunohistochemical analysis of paraffin-embedded KARPAS 299 cell pellet (left, high-expressing) or VCaP cell pellet (right, low-expressing) using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 44

Immunoprecipitation of SQSTM1/p62 protein from PANC-1 cell extracts. Lane 1 is 10% input, lane 2 is Mouse (G3A1) mAb IgG1 Isotype Control #5415, and lane 3 is SQSTM1/p62 (D5L7G) Mouse mAb. Western blot was performed using SQSTM1/p62 (D5L7G) Mouse mAb. A light chain-specific secondary antibody was used to avoid reactivity with heavy chain IgG.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 45

Western blot analysis of extracts from THP-1 cells, differentiated with TPA #4174 (50 ng/ml, overnight) and then treated with LPS #14011 (5 μg/ml, indicated times), using Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb (upper), total Gasdermin D (L60) Antibody #93709 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 46

Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, untreated (left) or treated with Chloroquine #14774 (right), using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 47

Immunohistochemical analysis of paraffin-embedded normal human spleen using LC3B (E5Q2K) Mouse mAb.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 48

Western blot analysis of extracts from various cell lines using SQSTM1/p62 (D5L7G) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 49

Immunoprecipitation of LC3B from HeLa cells treated with Chloroquine (50 μM, overnight) # 14774. Lane 1 is 10% input, lane 2 is Mouse (G3A1) mAb IgG1 Isotype Control, and lane 3 is LC3B (E5Q2K) Mouse mAb. Western blot was performed using LC3B (E5Q2K) Mouse mAb. Anti-mouse IgG, HRP-linked Antibody #7076 was used as a secondary antibody.

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 50

Western blot analysis of extract from HCT 116 cells, untreated (-) or starved with Earles Basic Salt Solution (EBSS; 4 hr; +) using SQSTM1/p62 (D5L7G) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 51

Western blot analysis of extracts from HeLa, C2C12, and KNRK cells, untreated (-) or treated with Chloroquine (50 μM, overnight; +) #14774 using LC3B (E5Q2K) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 52

Western blot anlaysis of extracts from HeLa cells, untreated (-) or treated with Chloroquine #14774 (50 μM, overnight; +) using SQSTM1/p62 (D5L7G) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 53

Western blot analysis of extracts from HeLa cells or HeLa cells with a knockout of LC3B (HeLa/LC3B KO) using LC3B (E5Q2K) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 54

Western blot analysis of extracts from HeLa cells transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® SQSTM1/p62 siRNA I #6394 (+), or SignalSilence® SQSTM1/p62 siRNA II #6399 (+), using SQSTM1/p62 (D5L7G) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 55

Western blot analysis of extracts from A549 cells, untreated (-) or starved with Earle's Balanced Salt Solution (EBSS) (indicated times) using LC3B (E5Q2K) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb (lower).

Human Reactive Cell Death and Autophagy Antibody Sampler Kit: Image 56

Western blot analysis of extracts from MCF7 and Raji cells, untreated (-) or treated with Torin 1 (250 nM, 5 hr; +) #14379, using LC3B (E5Q2K) Mouse mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

To Purchase # 42867T
Product # Size Price
42867T
1 Kit  (9 x 20 µl) $ 609

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb 9664 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R Mk 17, 19 Rabbit IgG
Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb 5625 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H Mk 89 Rabbit IgG
Phospho-RIP (Ser166) (D1L3S) Rabbit mAb 65746 20 µl
  • WB
H 78-82 Rabbit IgG
Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb 93654 20 µl
  • WB
  • IF
H 46-62 Rabbit IgG
Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb 91689 20 µl
  • WB
H 54 Rabbit IgG
Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb 36425 20 µl
  • WB
  • IP
  • IHC
H 30 Rabbit IgG
Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb 83186 20 µl
  • WB
  • IP
  • IF
H 17 Rabbit IgG
LC3B (E5Q2K) Mouse mAb 83506 20 µl
  • WB
  • IP
  • IHC
  • IF
H M R 14, 16 Mouse IgG2b
SQSTM1/p62 (D5L7G) Mouse mAb 88588 20 µl
  • WB
  • IP
  • IHC
  • IF
H 62 Mouse IgG1
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The Human Reactive Cell Death and Autophagy Antibody Sampler Kit provides an economical means of detecting common readouts in apoptosis, necroptosis, pyroptosis, and autophagy. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Each antibody in the Human Reactive Cell Death and Autophagy Antibody Sampler Kit detects endogenous levels of its target protein. Cleaved Caspase-3 (Asp175) (5A1E) Rabbit mAb detects endogenous levels of the large fragment (17/19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175. This antibody does not recognize full-length caspase-3 or other cleaved caspases. Non-specific labeling may be observed by immunofluorescence in specific sub-types of healthy cells in fixed-frozen tissues (e.g., pancreatic alpha-cells). Cytoplasmic background may be observed in human and monkey samples. Cleaved PARP (Asp214) (D64E10) XP® Rabbit mAb detects endogenous levels of the large fragment (89 kDa) of human PARP1 protein produced by caspase cleavage. The antibody does not recognize full-length PARP1 or other PARP isoforms. Cleaved Gasdermin D (Asp275) (E7H9G) Rabbit mAb recognizes endogenous levels of the amino-terminal fragment of Gasdermin D only when cleaved at Asp275. Cleaved-IL-1β (Asp116) (D3A3Z) Rabbit mAb recognizes endogenous levels of mature IL-1β protein only when cleaved at Asp116. Phospho-RIP3 (Ser227) (D6W2T) Rabbit mAb detected a band at 30 kDa that appears to be a cleavage product of RIP3. Phospho-MLKL (Ser358) (D6H3V) Rabbit mAb may also bind to MLKL when dually phosphorylated at Thr357 and Ser358. LC3B (E5Q2K) Mouse mAb detects both type I and type II forms of LC3B. Cross reactivity was not detected with other family members.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Asp175 of human caspase-3, Asp214 of human PARP, Asp275 of human Gasdermin D, Asp116 of human IL-1β, Pro220 of human SQSTM1/p62, residues near the amino terminus of human LC3B, and synthetic phosphopeptides corresponding to Ser166 of human RIP, Ser227 of human RIP3, and Ser358 of human MLKL.

Background

Regulated cell death has been classified based on distinct morphological and biochemical pathways (1). Type I cell death, or apoptosis, is characterized by cytoplasmic shrinkage, chromatin condensation, nuclear fragmentation, plasma membrane blebbing, and phagocytic update of dead cells. Apoptosis can occur through extrinsic pathways involving extracellular factors, including the activation of death receptors, or through intrinsic pathways involving intracellular perturbations, including mitochondrial outer membrane permeabilization (2). Both of these apoptotic pathways lead to activation of caspases, a family of cysteine acid proteases that are synthesized as inactive zymogens containing pro-domains, followed by large (p20) and small (p10) subunits which are proteolytically activated in a cascade-like fashion. Caspase-3 is a key downstream protease activated by both extrinsic and intrinsic apoptotic pathways and cleaves a large number of proteins involved in the disassembly of the cell, including poly(ADP-ribose) polymerase (PARP), a protein involved in the DNA damage response.

 

Type II cell death, or autophagy, manifests with extensive cytoplasmic vacuolization, and like apoptosis, can include phagocytic update. Autophagy is a catabolic process for the degradation of cellular components including protein aggregates, damaged organelles, and pathogens (3). The process involves the engulfment of these components into a double membrane structure, the autophagosome, which fuses to the lysosome for degradation. Autophagy requires, and can be monitored by, the conversion of LC3 family members, such as LC3B, from a type I form to a lipidated type II form that is incorporated into the autophagosome membrane and binds to a variety of cargo receptors. Cargo receptors such as SQSTM1/p62 bind LC3 along with ubiquitinated proteins that are targeted for degradation. SQSTM1/p62 is also degraded during this process, and thus its expression is frequently used to monitor this process.

Type III cell death, or necrosis, manifests with plasma membrane permeability with cellular swelling and fragmentation, and lacks a clear phagocytic response which then leads to an inflammatory signaling with the release of damage-associated molecular patterns (DAMPs). Necrosis can be triggered by multiple regulated pathways including necroptosis and pyroptosis. Necroptosis is regulated by the kinase activities of RIP and RIP3 and the pore forming ability of MLKL (4). Necroptosis requires the activation of RIP3 which then phosphorylates MLKL at Ser358 (Ser345 in mouse). Phosphorylation of MLKL leads to generation of a pore complex involved in cell swelling and the secretion of DAMPs. RIP3 activation is triggered through several RIP homotypic interaction motif (RHIM) domain interactions including RIP, TRIF, and ZBP1 and results in the phosphorylation of RIP3 at Ser227 (Thr231/Ser232 in mouse). Canonical necroptosis signaling is mediated by RIP, and this can be inhibited by necrostatins, small molecules that directly inhibit RIP kinase activity. Activation of RIP can be monitored through autophosphorylation sites including Ser166. Pyroptosis is generally induced in cells of the innate immune system, and is characterized by cleavage of Gasdermin D (5). The amino-terminal fragment of Gasdermin D produced following cleavage by inflammatory caspases (Caspase-1, -4, -5), oligomerizes to form a pore. Canonical cleavage of Gasdermin D occurs through a two-step process. The first step involves transcriptional regulation of targets such as NLRP3 and the pro-forms of IL-1β and IL-18. In the second execution step, Caspase-1 is activated through formation of inflammasome complexes. Activated Caspase-1 cleaves Gasdermin D as well as IL-1β and IL-18 to their mature forms, and these active cytokines are secreted through pores formed by Gasdermin D.

  1. Galluzzi, L. et al. (2018) Cell Death Differ 25, 486-541.
  2. Green, D.R. (1998) Cell 94, 695-8.
  3. Codogno, P. and Meijer, A.J. (2005) Cell Death Differ 12 Suppl 2, 1509-18.
  4. Shan, B. et al. (2018) Genes Dev 32, 327-40.
  5. Shi, J. et al. (2017) Trends Biochem Sci 42, 245-54.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
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