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48734
Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit #48734

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other Image 1 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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).

other Image 2 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Flow cytometric analysis of serum-starved Neuro-2a cells, untreated (blue) or treated with Staurosporine #9953 (1 μM, 3 hr; green), using Cleaved PARP (Asp214) (D6X6X) Rabbit mAb (Rodent Specific) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

other Image 3 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunoprecipitation of phospho-RIP (Ser166) protein from L-929 cell extracts treated with Z-VAD(OMe)-FMK #60332 (20 μM, 30 min) followed by Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/ml, 2 hr) and SM-164 (100 nM, 2 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 Phospho-RIP (Ser166) (E7G6O) Rabbit mAb. Western blot analysis was performed using Phospho-RIP (Ser166) (E7G6O) Rabbit mAb. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.

other Image 4 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Western blot analysis of extracts from wild-type (WT) MEF or MEF/RIPK1 knockout (KO) cells, untreated (-) or treated with Z-VAD(OMe)-FMK #60332 (20 μM, 30 min) followed by Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/ml, 7 hr) and SM-164 (100 nM, 7 hr), using Phospho-RIP (Ser166) (E7G6O) Rabbit mAb (upper), RIP (D94C12) XP® Rabbit mAb #3493 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). MEF/RIPK1 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston, MA.

other Image 5 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Confocal immunofluorescent analysis of L-929 cells, untreated (left), pre-treated with Z-VAD (20 μM, 30 min) followed by treatment with SM-164 (100 nM) and Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/mL, 2.25 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-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb (green). Samples were mounted in ProLong® Gold Antifade Reagent with DAPI #8961 (blue).

other Image 6 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Confocal immunofluorescent analysis of L-929 cells, untreated (left), pre-treated with Z-VAD (20 μM, 30 min) followed by treatment with SM-164 (100 nM) and Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/mL, 2.5 hr; center) and then post-processed with λ-phosphatase (right), using Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb (green). Red = Propidium Iodide (PI)/RNase Staining Solution #4087 (fluorescent DNA dye).

other Image 7 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Western blot analysis of extracts from mouse bone marrow derived macrophages (mBMDM), untreated (-) or treated with Lipopolysaccharides (LPS) #14011 (50 ng/ml, 4 hr; +) followed by Nigericin (sodium salt) #66419 (15 μM, indicated times; +), using Cleaved Gasdermin D (Asp276) (E3E3P) Rabbit mAb (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

other Image 8 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunoprecipitation of Cleaved-IL-1β (Asp117) from extracts of media from mouse bone marrow derived macrophages (mBMDM) treated with LPS #14011 (50 ng/ml, 4 hr) followed by nigericin (15 μM, 45 min). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Cleaved-IL-1β (Asp117) (E7V2A) Rabbit mAb (Mouse Specific). Western blot was performed using Cleaved-IL-1β (Asp117) (E7V2A) Rabbit mAb (Mouse Specific). Anti-Rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.

other Image 9 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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).

other Image 10 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Confocal immunofluorescent analysis of wild-type MEFs, either untreated (left) or treated with Chloroquine #14774 (50 μM, 18 hours; center), and SQSTM1/p62 knock-out MEFs treated with chloroquine (right), using SQSTM1 (D6M5X) Rabbit mAb (green). Actin filaments were labeled with β-Actin (8H10D10) Mouse mAb #3700 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye). MEF SQSTM1/p62 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston MA.

other Image 11 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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.

other Image 12 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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).

other Image 13 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Flow cytometric analysis of serum-starved H-4-II-E cells, untreated (blue) or treated with Staurosporine #9953 (1 μM, 3 hr; green), using Cleaved PARP (Asp214) (D6X6X) Rabbit mAb (Rodent Specific) (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

other Image 14 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Western blot analysis of extracts from L-929 cells, untreated (-) or treated with Z-VAD(OMe)-FMK #60332 (20 μM, 30 min) followed by Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/ml, indicated times) and SM-164 (100 nM, indicated times), using Phospho-RIP (Ser166) (E7G6O) Rabbit mAb (upper), RIP (D94C12) XP® Rabbit mAb #3493 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

other Image 15 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Western blot analysis of L-929 cells, untreated (-), or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), SM-164 (100 nM, 3 hr; +), and mouse TNF-α (20 ng/ml, 3 hr; +), using Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb (upper), RIP3 (D8J3L) Rabbit mAb #15828 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

other Image 16 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Western blot analysis of L-929 cells, untreated (-), or treated with combinations of the following treatments as indicated: Z-VAD (20 μM, added 30 min prior to other compounds; +), Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/ml, 4 hr; +), SM-164 (100 nM, 4 hr; +), and necrostatin-1 (Nec-1, 50 μM, 4 hr; +), using Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb (upper), total MLKL (D6W1K) Rabbit mAb (Mouse Specific) #37705 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

other Image 17 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Western blot analysis of cell extracts and media from mouse bone marrow derived macrophages (mBMDM), untreated (-), or treated (+) with combinations of LPS #14011 (50 ng/ml, 4 hr) followed by nigericin (15 μM, 45 min) using Cleaved-IL-1β (Asp117) (D7V2A) Rabbit mAb (Mouse Specific) (upper) or total IL-1β (D3H1Z) Rabbit mAb (Mouse Specific) #12507 (lower).

other Image 18 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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

other Image 19 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunohistochemical analysis of paraffin-embedded MEF wild-type cell pellet (left, positive) or MEF SQSTM1/p62 KO cell pellet (right, negative) using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific). MEF SQSTM1/p62 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston MA.

other Image 20 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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).

other Image 21 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Confocal immunofluorescent analysis of Neuro-2a cells, untreated (left, negative) or treated with Staurosporine #9953 (1 μM, 3 hr; right, positive), using Cleaved PARP (Asp214) (D6X6X) Rabbit mAb (Rodent Specific) (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

other Image 22 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Western blot analysis of extracts from H9c2(2-1) cells, untreated (-) or treated with Z-VAD(OMe)-FMK #60332 (20 μM, 30 min) followed by Mouse Tumor Necrosis Factor-α (mTNF-α) #5178 (20 ng/ml, 4.5 hr) and SM-164 (100 nM, 4.5 hr), using Phospho-RIP (Ser166) (E7G6O) Rabbit mAb (upper), RIP (D94C12) XP® Rabbit mAb #3493 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

other Image 23 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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

other Image 24 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunohistochemical analysis of paraffin-embedded mouse forestomach using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific).

other Image 25 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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)).

other Image 26 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunohistochemical analysis of paraffin-embedded 3T3 cell pellet, untreated (left, negative) or treated with Staurosporine #9953 (right, positive), using Cleaved PARP (Asp214)(D6X6X) mAb (Rodent Specific).

other Image 27 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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

other Image 28 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunohistochemical analysis of paraffin-embedded mouse kidney using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific).

other Image 29 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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

other Image 30 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunohistochemical analysis of paraffin-embedded E14 rat embryo using Cleaved PARP (Asp214)(D6X6X) mAb (Rodent Specific).

other Image 31 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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

other Image 32 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunohistochemical analysis of paraffin-embedded mouse spleen using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific).

other Image 33 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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.

other Image 34 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunohistochemical analysis of paraffin-embedded H-4-II-E cell pellet, untreated (left, negative) or treated with Staurosporine #9953 (right, positive), using Cleaved PARP (Asp214)(D6X6X) mAb (Rodent Specific).

other Image 35 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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.

other Image 36 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunohistochemical analysis of paraffin-embedded rat spleen using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific).

other Image 37 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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.

other Image 38 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunohistochemical analysis of paraffin-embedded mouse ovary using Cleaved PARP (Asp214)(D6X6X) mAb (Rodent Specific).

other Image 39 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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).

other Image 40 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunohistochemical analysis of paraffin-embedded mouse small intestine using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific).

other Image 41 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunohistochemical analysis of paraffin-embedded mouse spleen using Cleaved PARP (Asp214)(D6X6X) mAb (Rodent Specific).

other Image 42 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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).

other Image 43 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunoprecipitation of SQSTM1 from L-929 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific). Western blot was performed using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific). Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.

other Image 44 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Immunoprecipitation of Cleaved PARP (Asp214) from Neuro-2a cell extracts treated with Staurosporine #9953 (1 μM, 3 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Cleaved PARP (Asp214) (D6X6X) Rabbit mAb (Rodent Specific). Western blot was perform using Cleaved PARP (Asp214) (D6X6X) Rabbit mAb (Rodent Specific). Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.

other Image 45 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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).

other Image 46 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Western blot analysis of extracts from MEFs from wild-type or SQSTM1/p62 knockout mice using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). MEF SQSTM1/p62 KO cells were kindly provided by Dr. Junying Yuan, Harvard Medical School, Boston MA.

other Image 47 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Western blot analysis of extracts from serum-starved Neuro-2a cells, untreated (-) or treated with Staurosporine #9953 (1 μM, 3 hr), using Cleaved PARP (Asp214) (D6X6X) Rabbit mAb (Rodent Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

other Image 48 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

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).

other Image 49 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Western blot analysis of extracts from C2C12 cells, untreated (-) or treated with Chloroquine #14774 (50 μM, overnight) using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

other Image 50 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Western blot analysis of extracts from serum-starved H-4-II-E cells, untreated (-) or treated with Staurosporine #9953 (1 μM, 6 hr; +), using Cleaved PARP (Asp214) (D6X6X) Rabbit mAb (Rodent Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

other Image 51 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Western blot analysis of extracts from various cell lines using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific).

other Image 52 - Mouse Reactive Cell Death and Autophagy Antibody Sampler Kit

Western blot analysis of extracts from MEFs, untreated (-) or treated with Earles Basic Salt Solution (EBSS; 4 hr; +) using SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific) (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

To Purchase # 48734T
Product # Size Price
48734T
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) (D6X6X) Rabbit mAb (Rodent Specific) 94885 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
M R 89 Rabbit IgG
Phospho-RIP (Ser166) (E7G6O) Rabbit mAb 53286 20 µl
  • WB
  • IP
M R 78 Rabbit IgG
Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb 91702 20 µl
  • WB
  • IF
M 46-62 Rabbit IgG
Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb 37333 20 µl
  • WB
  • IF
M 54 Rabbit IgG
Cleaved Gasdermin D (Asp276) (E3E3P) Rabbit mAb 10137 20 µl
  • WB
M 31 Rabbit IgG
Cleaved-IL-1β (Asp117) (E7V2A) Rabbit mAb (Mouse Specific) 63124 20 µl
  • WB
  • IP
M 17 Rabbit IgG
LC3B (E5Q2K) Mouse mAb 83506 20 µl
  • WB
  • IP
  • IHC
  • IF
H M R 14, 16 Mouse IgG2b
SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific) 23214 20 µl
  • WB
  • IP
  • IHC
  • IF
M R 62 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Product Description

The Mouse 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 Mouse 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. Cleaved PARP (Asp214) (D6X6X) Rabbit mAb (Rodent Specific) recognizes endogenous levels of the large fragment (89 kDa) of rodent PARP only when cleaved at Asp214. Cleaved Gasdermin D (Asp276) (E3E3P) Rabbit mAb recognizes endogenous levels of the amino fragment of mouse Gasdermin D protein only when cleaved at Asp276. Cleaved-IL-1β (Asp117) (E7V2A) Rabbit mAb (Mouse Specific) recognizes endogenous levels of mouse IL-1β protein only when cleaved at Asp117. Phospho-RIP3 (Thr231/Ser232) (E7S1R) Rabbit mAb recognizes endogenous levels of RIP3 protein only when phosphorylated at Thr231/Ser232. This antibody may not recognize RIP3 when only singly phosphorylated at Thr231 or Ser232. Phospho-RIP (Ser166) (E7G6O) Rabbit mAb recognizes endogenous levels of RIP protein only when phosphorylated at Ser166. Phospho-MLKL (Ser345) (D6E3G) Rabbit mAb recognizes endogenous levels of mouse MLKL protein only when phosphorylated at Ser345. Weak, non-specific nuclear staining has been observed by immunofluorescence (IF-IC). LC3B (E5Q2K) Mouse mAb detects both type I and type II forms of LC3B. Cross reactivity was not detected with other family members. SQSTM1/p62 (D6M5X) Rabbit mAb (Rodent Specific) recognizes endogenous levels of total rodent SQSTM1/p62 protein.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Asp175 of human caspase-3, Asp214 of rodent PARP, Asp276 of mouse Gasdermin D, Asp117 of mouse IL-1β, Gly300 of mouse SQSTM1/p62, residues near the amino terminus of human LC3B, and synthetic phosphopeptides corresponding to Ser166 of mouse RIP, Thr231/Ser232 of mouse RIP3, and Ser345 of mouse 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.

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