Mitophagy Antibody Sampler Kit

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Mitophagy Antibody Sampler Kit

品牌： CST

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产品介绍

产品信息

荧光素标记

Unconjugated

抗原名称

Mitophagy

简单描述

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

研究领域

癌症,细胞生物学,纤维化,免疫学和肿瘤学,代谢,神经科学

背景

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 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). 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.

研究领域

癌症,细胞生物学,纤维化,免疫学和肿瘤学,代谢,神经科学

翻译后修饰

phosphate

数据库链接

Entrez-Gene ID

664,65018,8878,10133,10241,7311,81631,7316,5071,7314,6233,665

UniProt ID

Q12983,Q9BXM7,Q13501,Q96CV9,Q13137,P62987,Q9GZQ8,P0CG48,O60260,P0CG47,P62979,O60238

参考图片

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.

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).

Western blot analysis of extracts from HCT 116 and HCT 116 LC3B knockout cells, untreated (-) or treated with Chloroquine #14774 (50 μM, 18 hr) using LC3B (D11) XP® Rabbit mAb #3868 (upper) or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). The absence of signal in the HCT 116 knockout cells confirms the specificity of the antibody for LC3B.

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

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).

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

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

Western blot analysis of extracts from PC-3 cells, untreated (-) or treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 30 μM, 6 hr; +), using Phospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAb (upper), PINK1 (D8G3) Rabbit mAb #6946 (middle), or GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

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.

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.

Western blot analysis of extracts from HeLa cells (lane 1) or SQSTM1 knock-out cells (lane 2) using SQSTM1/p62 (D5E2) Rabbit mAb #8025 (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.

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

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.

Immunoprecipitation of Parkin protein from PC12 extracts. Lane 1 is 10% input, lane 2 is Mouse (E7Q5L) mAb IgG2b Isotype Control #53484, and lane 3 is Parkin (Prk8) Mouse mAb. Western blot analysis was performed using Parkin Antibody #2132. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.

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.

Western blot analysis of extracts from PC-3 cells treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP; 30 μM, 6 hr ). Lysates were treated with (+) or without (-) Lambda Phosphatase (λ-Phosphatase) and Calf Intestinal Phosphatase (CIP). Western blot was performed using Phospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

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

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.

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.

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.

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 Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Phospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAb. Western blot was performed using Phospho-Ubiquitin (Ser65) (E2J6T) Rabbit mAb. Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (HRP Conjugate) #5127 was used as a secondary antibody.

Western blot analysis of HAP1 extracts from WT or PINK1 KO untreated (-) or treated with CCCP (+) using PINK1 (D8G3) Rabbit mAb. Membranes stained with Ponceau S for total protein normalization (lower). These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibodies, as a companion to validation data generated by CST scientists.

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.

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

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).

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

Confocal immunofluorescent analysis of HCT 116 cells either untreated (left) or treated with Chloroquine #14774 (50 µM, overnight) (center) or LC3B HCT 116 knockout cells treated with Chloroquine #14774 (50 µM, overnight) (right) using LC3B (D11) XP® Rabbit mAb (green). Actin filaments were labeled with β-Actin (8H10D10) Mouse mAb (red) and nuclei were labeled with DAPI #4083 (blue).

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

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).

Flow cytometric analysis of HCT-116 cells, wild-type (green, high expression) or LC3B knockdown (blue, negative expression), using LC3B (D11) XP® Rabbit mAb #3868 (solid lines) or a 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.

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).

Immunoprecipitation of PINK1 protein from PC-3+CCCP extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control, #3900 and lane 3 is PINK1 (D8G3) Rabbit mAb. Western blot analysis was performed using PINK (D8G3) Rabbit mAb, #6946. Anti-rabbit IgG, HRP-linked Antibody, #7074 was used as the secondary antibody.

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

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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货号：

43110T

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