Whole cell lysates (50 μg) were separated by SDS-PAGE and the proteins transferred to a nitrocellulose membrane for Western blotting as previously described (20 (link)). Specific primary antibodies were prepared according to the manufacturer’s instructions; Cyclin D1 (Cat. No. sc-753, clone H295, Santa Cruz, Santa Cruz, CA, USA), phospho Rb S780 (Cat. No. 9307S, Cell Signaling, Beverly, MA, USA), LC3-A/B (Cat. No. 4108, Cell Signaling, Danvers, MA, USA), AMPK (Cat. No. 2532S, Cell Signaling, Beverly, MA, USA), phospho AMPK T172 (Cat. No. 2531S, Cell Signaling, Beverly, MA, USA), Bnip3 (Cat. No. 3485-1, Epitomics, Burlingame, CA, USA) and Vinculin (Cat. No. V9131, Sigma-Aldrich, St. Louis, USA), which was used as an internal protein loading control. The LKB1 phospho-serine 325 (pS325) antibody was previously described (6 (link)). Anti rabbit (Cat. No. sc-2004, Santa Cruz, Santa Cruz, CA, USA) or mouse (Cat. No. sc-2005, Santa Cruz, Santa Cruz, CA, USA) secondary HRP-conjugated antibodies were diluted at 1:3000.
Bnip3
Manufactured by Cell Signaling Technology
Sourced in United States
BNIP3 is a protein that plays a role in the process of autophagy, which is the degradation and recycling of cellular components. It is involved in the formation of autophagosomes, which are the structures that engulf and deliver cellular material to the lysosome for degradation. BNIP3 is an important regulator of this cellular process.
Automatically generated - may contain errors
Lab products found in correlation
Beclin 1, by Cell Signaling Technology (9 mentions)
Parkin, by Cell Signaling Technology (8 mentions)
β actin, by Cell Signaling Technology (4 mentions)
Hif 1α, by Cell Signaling Technology (4 mentions)
Cox 4, by Cell Signaling Technology (4 mentions)
Bcl 2, by Cell Signaling Technology (4 mentions)
Lc3a b, by Cell Signaling Technology (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Lc3 2, by Cell Signaling Technology (3 mentions)
Caspase 9, by Cell Signaling Technology (3 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (3 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Ubiquitin, by Cell Signaling Technology (2 mentions)
P jnk, by Cell Signaling Technology (2 mentions)
Cleaved caspase 9, by Cell Signaling Technology (2 mentions)
Caspase 3, by Cell Signaling Technology (2 mentions)
Cytochrome c, by Cell Signaling Technology (2 mentions)
P ampk, by Cell Signaling Technology (2 mentions)
P ulk1, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
31 protocols using bnip3
1
Western Blot Analysis of Cell Signaling Proteins
2
Western Blot Analysis of Cellular Proteins
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Lysates from cells and tissues were subjected to 10 % SDS-PAGE (Bio-Rad, Pre-gel, MA); separated proteins were transferred onto Polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Seoul, Korea). Membranes were blocked with TBS-based blocking buffer (Li-COR Biosciences, Lincoln, NE) for 1h at room temperature, and incubated overnight at 4 °C with primary antibodies p16, SOD1,SOD2, ERK, p-ERK, CREB, p-CREB, p-elF2α, Beclin1, Bip, ATG12, LC3B, p62, BNIP3, PINK1, Parkin, PPARα, FATP4, Tau, P-tau, Beta-amyloid, GFAP, iba-1, beta-actin (1:1000, Cell Signaling Technology, Danvers, MA). Membranes were then washed with TBST three times for 10 min each and incubated with horseradish peroxidase-conjugated secondary antibodies (1:3000, Cell Signaling Technology, Danvers, MA) for 1 h at room temperature and washed again. Signals were detected by Odyssey-LC chemiluminsescent imaging system (LI-COR, Lincoln, NE). Signals were averaged and expressed as described in figure legend.
3
Western Blot Analysis of Autophagy and Apoptosis Markers
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Total protein extracts were prepared from SH-SY5Y cells using radioimmunoprecipitation assay (RIPA) buffer. Equal amounts of protein samples (20–40 μg) were resolved on SDS-PAGE gels and transferred onto Immobilon polyvinyl difluoride membranes (GE Healthcare, Little Chalfont, UK) using standard protocols. The membranes were blocked with 4% bovine serum albumin (BSA) for 1 h at room temperature and probed with primary antibody against HIF-1α (1:2000; Cell Signaling Technology), BNIP3 (1:1500; Cell Signaling Technology), Beclin1 (1:1000; Abcam, Cambridge, MA, USA), LC3 (1:1000; Cell Signaling Technology), p62 (1:1000; Abcam), cleaved caspase-3 (1:2000; Cell Signaling Technology), GAPDH (1:2000; Cell Signaling Technology), or β-actin (1:1000; Beyotime Biotechnology, Haimen, China) at 4 °C overnight. After being washed with TBST (a mixture of tris-buffered saline and polysorbate 20 ), the blots were incubated with secondary goat anti-mouse or anti-rabbit antibody conjugated with horseradish peroxidase at room temperature for 2 h. After being washed three times, the proteins were visualized using an enhanced chemiluminescence kit (GE Healthcare). The proteins were quantified with ImageJ software and normalized to β-actin in parallel experiments.
4
Western Blot Analysis of Cardiac Proteins
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1 μg left ventricular myocardial tissues excluded infarct area or 10ˆ6 H9C2 cells were cut into small pieces and treated with radioimmunoprecipitation assay (RIPA) buffer containing 1% SDS and protease inhibitors. Then, the lysate was subjected to SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories). The membrane was blocked with 5% fat-free milk in PBST for 30 min, followed by incubation overnight at 4°C with final dilutions of primary antibodies against HIF-1α (#14179, Cell Signaling Technology, Beverly, MA, USA), VEGFA (ab231260, Abcam, Cambridge, MA, USA), GLUT1 (21829-1, Proteintech Group, Rosemont, IL, USA), LDHA (#3558, Cell Signaling Technology), BNIP3 (#3769, Cell Signaling Technology), ADRB2 (#13096-1, Proteintech Group), NCBP3 (ab91556, Abcam), EWSR1 (#55191-1, Proteintech Group), DRG2 (#14743-1, Proteintech Group), eIF4A2 (#11280-1, Proteintech Group), and GAPDH (#60004-1, Proteintech Group). After that, the membrane was washed three times and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Proteintech Group). The blotting bands were developed with ECL plus immunoblotting detection reagents (Thermo Fisher Scientific) and captured using ImageJ.
5
Protein Expression Analysis via Western Blot
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Western blot analysis was performed as depicted in a previous study (Serna-Salas et al., 2018 (link)). The primary antibodies (GAPDH, Mfn2, and P62 from Abcam; Beclin1, Atg5, and BNIP3 from Cell Signaling Technology; ATP synthase β and LC3 from Sigma) are listed in Supplementary Table S2
6
Mitochondrial Dynamics in Skeletal Muscle
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Western blot analysis of the gastrocnemius and EDL muscles were completed as we have described.21 Antibodies included COX‐IV (Cell Signaling 4844S), PGC‐1α (Novus Biologicals, NBP1‐04676SS), MFN1 (Santa Cruz sc‐50330), MFN2 (Santa Cruz sc‐50331), OPA1 (Santa Cruz sc‐367890), DRP1 (Cell Signaling 14647), FIS1 (Novus NB100‐56646). BNIP3 (Cell Signaling 3769), LC3 (Cell Signaling 4108), p62 (Cell Signaling 5114s). Western blot analysis was only performed in gastrocnemius and EDL tissues due to tissue availability.
7
Immunoblotting, Immunoprecipitation, and Immunohistochemistry Antibodies
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The antibodies used for immunoblotting, immunoprecipitation and immunohistochemistry were: PERM1 (Sigma, #HPA031711), diluted 1:1,000 for immunoblotting, 1:100 for immunohistochemistry; BNIP3 (Cell Signaling, #3769), diluted 1:1,000; TOM20 (Abcam, #ab56783), diluted 1:100 for immunohistochemistry; TOM20 (Sigma, #HPA011562), diluted 1:5,000 for immunoblotting, 1:100 for immunohistochemistry; GAPDH (Invitrogen, #AM4300), diluted 1:10,000 for immunoblotting; Ankyrin B (Invitrogen, #33-3700), diluted 1:1000 for immunoblotting, 1:100 for immunohistochemistry; MYH7 (Developmental Studies Hybridoma Bank [DSHB], BA-F8), MYH2 (DSHB, SC-71), MYH4 (DSHB, BF-F3), CD31/PECAM (BD Pharmingen, #553370), all diluted 1:100 for immunohistochemistry; anti-FLAG M2-HRP (Sigma, #A8592), anti-HA-HRP (Miltenyi, #130-091-972), Alexa Fluor 350 anti-mouse IgG2b (Life Technologies, #A-21140), Alexa Fluor 488 anti-mouse IgG1 (Life Technologies, #A-21121), Alexa Fluor 546 anti-mouse IgM (Life Technologies, #A-21045), Alexa Fluor 546 anti-rabbit (H+L) (Life Technologies, #A-11010), Alexa Fluor 488 anti-rat (Life Technologies, #A-11006), anti-mouse HRP (Sigma, #A9044) and anti-rabbit HRP (Sigma, #A0545), all diluted 1:4,000 for immunoblotting and 1:200 for immunohistochemistry.
8
Western Blot Analysis of Cellular Signaling
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Cells were lysed in Laemmli buffer (0.125 M Tris-HCl, pH 6.8, 2% SDS, 5% β-Mercaptoethanol, 10% glycerine, 0.002% bromophenol blue and 1% protease inhibitor cocktail (APExBIO, K1007)). The lysates were separated on 10% or 12% (wt/vol) SDS-polyacrylamide gels. After SDS/PAGE, proteins were transferred to PVDF membrane for Western blotting in transfer buffer with 10% (vol/vol) methanol at 4°C followed by standard Western-blotting protocols. β-actin or α-Tubulin was used for normalizing the protein load. All blots were repeated at least three times. Comparisons of relative levels of specific antigens were done by quantitative densitometry using ImageJ Software (version 1.48). Antibodies: HIF1α (Cell Signaling Technology, 36169), BNIP3 (Cell Signaling Technology, 3769), Ubiquitin (Cell Signaling Technology, 3936), P70S6K (Cell Signaling Technology, 2708), p-P70S6K (Cell Signaling Technology, 9234), PDK1 (Abcam, ab207450), mTOR (Abcam, ab134903), p-mTOR (Abcam, ab109268), AKT1/2/3 (Abcam, ab179463), p-AKT1/2/3 (Abcam, ab192623), LC3II (Zen BioScience, 306019), β-actin (Zen BioScience, 700068), α-Tubulin (Beyotime, AF0001), peroxidase-conjugated anti-rabbit (Beyotime, A0216) and peroxidase-conjugated anti-mouse antibodies (Beyotime, A0216).
9
Quantifying Mitophagy via Fluorescence Microscopy
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Cells were first fixed with 4% paraformaldehyde for 30 min at room temperature. After incubation with 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity, the samples were treated with the primary antibodies at 4 °C overnight. After the slides were washed with PBS and then incubated with secondary antibody (1:500, Invitrogen, Carlsbad, CA, USA) at room temperature for 45 min. Nuclei were stained using DAPI. Images were observed with fluorescence microscopy (Olympus BX-61). The primary antibodies used in the present study were as follows: p-ERK (1:1000, Abcam, #ab176660), Bnip3 (1:1000, Cell Signalling Technology, #44060), Tom20 (mitochondria marker, 1:1000, Abcam, #ab186735), LAMP1 (lysosome marker, 1:1000, Abcam, #ab24170), cyt-c (1:1000; Abcam; #ab90529). Mitophagy is the result of fusion between mitochondria and lysosome. The green mitochondria locate with red lysosome would generate the orange mitophagy. Then, the number of orange dot was measured to quantify the number of mitophagy.
10
Signaling Pathway Regulation Protocol
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Anti-TSC2, anti-STAT3 phospho-Ser727 and Tyr705, Ref-1, Rel-A phospho-Ser365, anti-β-actin, VEGF-A, BNIP3, and anti-rpS6 phospho-Ser235/236 were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-HIF-1α was purchased from BD Transduction Laboratories (Oxford, UK). Rapamycin, FL3331, and JSH23 were bought from Merck (Darmstadt, Germany), and KU-0063794 was purchased from Chemquest Ltd. (Cheshire, UK), while APX3330, APX2009, APX2011, and RN7-58 were obtained from Apexian Pharmaceuticals (Indianapolis, IN, USA). Unless stated otherwise, all other lab chemicals were obtained from Merck.
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