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43110
Mitophagy Antibody Sampler Kit

Mitophagy Antibody Sampler Kit #43110

Western Blotting Image 1

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 (D5E2) Rabbit mAb (upper) or α-Tubulin (11H10) Rabbit mAb #2125 (lower). The SQSTM1/p62 (D5E2) Rabbit mAb confirms silencing of SQSTM1/p62 expression, while the α-Tubulin (11H10) Rabbit mAb is used as a loading control.

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Western Blotting Image 2

Western blot analysis of extracts from various cell lines using NDP52 (D1E4A) Rabbit mAb.

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Western Blotting Image 3

Western blot analysis of extracts from various cell lines using Optineurin (D2L8S) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 4

Western blot analysis of extracts from PC12 cells, fetal rat brain and mouse brain, using Parkin (Prk8) Mouse mAb.

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Western Blotting Image 5

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a cDNA construct expressing full-length human PINK1 (hPINK1, +) using PINK1 (D8G3) Rabbit mAb.

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Western Blotting Image 6

Western blot analysis of extracts from PC-3 cells, untreated (-) or treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (30 μM, indicated times; +), using Phospho-Ubiquitin (Ser65) Antibody (upper), PINK1 (D8G3) Rabbit mAb #6946 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 7

Western blot analysis of extracts from HeLa or MCF7 cells, untreated (-) or cobalt chloride-treated (100 μM, overnight; +), using BNIP3 (D7U1T) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 8

Western blot analysis of A172 and HeLa cells, untreated (-) or cobalt chloride-treated (100 μM, overnight; +), using BNIP3L/Nix (D4R4B) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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Western Blotting Image 9

Western blot analysis of extracts from various cell lines, untreated (-) or treated overnight with chloroquine (50 μM) (+), using LC3B (D11) XP® Rabbit mAb (upper) or LC3B Antibody #2775 (lower).

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Western Blotting Image 10

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.

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Western Blotting Image 11

Western blot analysis of extracts from various cell lines using SQSTM1/p62 (D5E2) Rabbit mAb.

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Western Blotting Image 12

Western blot analysis of extracts from HeLa cells, untreated (-) or treated with CCCP (10 μM, 24 hr; +), using PINK1 (D8G3) Rabbit mAb.

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IP Image 13

Immunoprecipitation of phospho-ubiquitin (Ser65) from extracts of PC-3 cells treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (30 μM, 6 hr). Lane 1 is 10% input, lane 2 is precipitated with Normal Rabbit IgG #2729, and lane 3 is Phospho-Ubiquitin (Ser65) Antibody. Western blot was performed using Phospho-Ubiquitin (Ser65) Antibody.

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Western Blotting Image 14

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc-tagged full-length human BNIP3 protein (hBNIP3-Myc; +), using BNIP3 (D7U1T) Rabbit mAb.

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Western Blotting Image 15

Western blot analysis of extracts from various cell lines using BNIP3L/Nix (D4R4B) Rabbit mAb.

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Western Blotting Image 16

Western blot analysis of extracts from HeLa cells, transfected with 100 nM SignalSilence® Control siRNA (Unconjugated) #6568 (-), SignalSilence® LC3B siRNA I #6212 (+) or SignalSilence® LC3B siRNA II #6213 (+), using LC3B (D11) XP® Rabbit mAb #3868 and α-Tubulin (11H10) Rabbit mAb #2125. The LC3B (D11) XP® Rabbit mAb confirms silencing of LC3B expression, while the α-Tubulin (11H10) Rabbit mAb is used to control for loading and specificity of LC3B siRNA.

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Western Blotting Image 17

Western blot analysis of extracts from SK-MEL-2 cells, untreated (-) or starved overnight in Earle's Balanced Salt Solution (EBSS) (+), using SQSTM1/p62 (D5E2) Rabbit mAb.

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IP Image 18

Immunoprecipitation of BNIP3 from MCF7 cells treated with cobalt chloride (100 μM; overnight). Lane 1 represents 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is BNIP3 (D7U1T) Rabbit mAb. Western blot analysis was performed using BNIP3 (D7U1T) Rabbit mAb. A conformation-specific secondary antibody was used to avoid reactivity with IgG.

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Western Blotting Image 19

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing DYKDDDDK-tagged full-length human BNIP3L/Nix (hBNIP3L/Nix-DYKDDDDK; +), using BNIP3L/Nix (D4R4B) Rabbit mAb. The BNIP3L/Nix construct was kindly provided by Dr. Reuben Shaw, Salk Institute for Biological Studies, La Jolla, CA.

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IHC-P (paraffin) Image 20

Immunohistochemical analysis of paraffin-embedded human astrocytoma using LC3B (D11) XP® Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

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Western Blotting Image 21

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a tagged human SQSTM1/p62 construct (+), using SQSTM1/p62 (D5E2) Rabbit mAb.

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IHC-P (paraffin) Image 22

Immunohistochemical analysis of paraffin-embedded human non-small cell lung carcinoma using BNIP3 (D7U1T) Rabbit mAb.

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IP Image 23

Immunoprecipitation of BNIP3L/Nix from A172 cells, treated with cobalt chloride (100 μM, overnight), using Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (lane 2) or BNIP3L/Nix (D4R4B) Rabbit mAb (lane 3). Lane 1 is 10% input. Western blot analysis was performed using BNIP3L/Nix (D4R4B) Rabbit mAb.

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IHC-P (paraffin) Image 24

Immunohistochemical analysis of paraffin-embedded HeLa cell pellets, control (left) or chloroquine-treated (right), using LC3B (D11) XP® Rabbit mAb.

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IHC-P (paraffin) Image 25

Immunohistochemical analysis of paraffin-embedded human ovarian carcinoma using BNIP3 (D7U1T) Rabbit mAb.

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IF-IC Image 26

Confocal immunofluorescent analysis of A172 cells, untreated (left) or cobalt chloride-treated (100 μM, overnight; right), using BNIP3L/Nix (D4R4B) Rabbit mAb (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Flow Cytometry Image 27

Flow cytometric analysis of HeLa cells using LC3B (D11) XP® Rabbit mAb (blue) compared to a nonspecific negative control antibody (red).

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IHC-P (paraffin) Image 28

Immunohistochemical analysis of paraffin-embedded MCF7 cell pellets, untreated (left) or cobalt chloride-treated (right), using BNIP3 (D7U1T) Rabbit mAb.

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IF-IC Image 29

Confocal immunofluorescent analysis of HCT-116 cells, untreated (left) or choroquine-treated (50 uM, overnight; right) using LC3B (D11) XP® Rabbit mAb (green) and β-Catenin (L54E2) Mouse mAb (Alexa Fluor® 555 Conjugate) #5612 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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IHC-P (paraffin) Image 30

Immunohistochemical analysis of paraffin-embedded normal human liver using BNIP3 (D7U1T) Rabbit mAb in the presence of control peptide (left) and antigen-specific peptide (right).

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IF-IC Image 31

Confocal immunofluorescent analysis of HeLa cells, untreated (left) or treated with cobalt chloride (100 μM, 24 hr; right), using BNIP3 (D7U1T) Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalliodin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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Product Includes Quantity Applications Reactivity MW(kDa) Isotype
SQSTM1/p62 (D5E2) Rabbit mAb 8025 20 µl
  • WB
  • IP
H Mk 62 Rabbit IgG
NDP52 (D1E4A) Rabbit mAb 60732 20 µl
  • WB
H 52, 60 Rabbit IgG
Optineurin (D2L8S) Rabbit mAb 58981 20 µl
  • WB
H 75 Rabbit IgG
Parkin (Prk8) Mouse mAb 4211 20 µl
  • WB
  • IP
H M R 50 Mouse IgG2b
PINK1 (D8G3) Rabbit mAb 6946 20 µl
  • WB
  • IP
H 60, 50 Rabbit IgG
Phospho-Ubiquitin (Ser65) Antibody 37642 20 µl
  • WB
  • IP
H Rabbit 
BNIP3 (D7U1T) Rabbit mAb 44060 20 µl
  • WB
  • IP
  • IHC
  • IF
H 22-28, 50-55 Rabbit IgG
BNIP3L/Nix (D4R4B) Rabbit mAb 12396 20 µl
  • WB
  • IP
  • IF
H M R Mk 38, 76 Rabbit IgG
LC3B (D11) XP® Rabbit mAb 3868 20 µl
  • WB
  • IP
  • IHC
  • IF
  • F
H M R 14, 16 Rabbit IgG
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 

Each antibody in the Mitophagy Antibody Sampler kit detects endogenous levels of its target protein. LC3B (D11) XP® Rabbit mAb detects type I and type II forms of LC3B. Weaker reactivity is observed with rodent LC3B.

Monoclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to residues surrounding Gly162 of human SQSTM1/p62, Val135 of human NDP52, Pro140 of human PINK1, Leu410 of human Optineurin, Glu128 of human BNIP3L/Nix, peptide sequences corresponding to residues near the amino terminus of human LC3B, the amino terminus of human BNIP3, and a recombinant fusion protein with an epitope that maps to the carboxy terminus of human Parkin. Polyclonal antibodies are produced by immunizing animals with a synthetic phospho-peptide corresponding to residues surrounding Ser65 of human Ubiquitin. Antibodies are purified by protein A and peptide affinity chromatography.

Autophagy is a catabolic process for the autophagosome-lysosomal degradation of bulk cytoplasmic contents (1, 2). Selective autophagy targets the degradation of distinct sets of substrates and organelles (3-5). One of the best studied examples of selective autophagy involves the clearance of damaged mitochondria through a process called mitophagy. Several pathways have been described for various contexts of mitophagy, including the FUNDC1 pathway, the BNIP3 and BNIP3L/Nix pathway, and the PINK1/Parkin pathway. FUNDC1 is a mitochondrial protein that is phosphorylated by the autophagy kinase ULK1 and regulates hypoxia induced mitophagy (6, 7). BNIP3L/Nix and BNIP3 are members of the Bcl-2 family of apoptosis regulators that are expressed on mitochondria, induced by hypoxia, and have have been shown to play a role in mitophagy (8). BNIP3L/Nix is also important in the autophagic maturation of erythroid cells (9). FUNDC1, BNIP3 and BNIP3L/Nix bind to LC3 family members, targeting the mitochondria to the autophagosome.

Non-hypoxic induction of mitophagy can be regulated by the PINK1/Parkin pathway, which plays causative roles in neurodegenerative disease, most notably Parkinson’s disease (10, 11). PINK1 is a mitochondrial serine/threonine kinase that is stabilized on the outer mitochondrial membrane of damaged mitochondria. Substrates of PINK1 include the E3 ubiquitin ligase Parkin and ubiquitin itself (12-14). Phosphorylation of Parkin as well as binding to phosphorylated ubiquitin leads to accumulation of ubiquitinated chains on multiple mitochondrial proteins. Ubiquitinated proteins are recognized by selective cargo receptors including SQSTM1/p62, Optineurin, and NDP52 (15-16). Autophagy cargo receptors contain an LC3-interacting region (LIR) required for binding to Atg8/LC3 family members and targeting to the autophagosome (3).

The Mitophagy Antibody Sampler Kit provides an economical means of detecting proteins involved in the process of mitophagy. The kit includes enough antibody to perform two western blot experiments with each primary antibody.

  1. Reggiori, F. and Klionsky, D.J. (2002) Eukaryot Cell 1, 11-21.
  2. Codogno, P. and Meijer, A.J. (2005) Cell Death Differ. 12 Suppl 2, 1509-18.
  3. Birgisdottir, Å.B. et al. (2013) J Cell Sci 126, 3237-47.
  4. Xu, Z. et al. (2015) Acta Biochim Biophys Sin (Shanghai) 47, 571-80.
  5. Mancias, J.D. and Kimmelman, A.C. (2016) J Mol Biol 428, 1659-80.
  6. Liu, L. et al. (2012) Nat Cell Biol 14, 177-85.
  7. Wu, W. et al. (2014) EMBO Rep 15, 566-75.
  8. Sowter, H.M. et al. (2001) Cancer Res 61, 6669-73.
  9. Sandoval, H. et al. (2008) Nature 454, 232-5.
  10. Kitada, T. et al. (1998) Nature 392, 605-8.
  11. Valente, E.M. et al. (2004) Science 304, 1158-60.
  12. Kim, Y. et al. (2008) Biochem Biophys Res Commun 377, 975-80.
  13. Kane, L.A. et al. (2014) J Cell Biol 205, 143-53.
  14. Koyano, F. et al. (2014) Nature 510, 162-6.
  15. Heo, J.M. et al. (2015) Mol Cell 60, 7-20.
  16. Lazarou, M. et al. (2015) Nature 524, 309-314.
Entrez-Gene Id
664 , 665 , 81631 , 10241 , 10133 , 5071 , 65018 , 6233 , 8878 , 7311 , 7314 , 7316
Swiss-Prot Acc.
Q12983 , O60238 , Q9GZQ8 , Q13137 , Q96CV9 , O60260 , Q9BXM7 , P62979 , Q13501 , P62987 , P0CG47 , P0CG48
For Research Use Only. Not For Use In Diagnostic Procedures.

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.

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