The following primary antibodies were used for Western blot (WB) and/or immunofluorescence (IF): rabbit anti-CDC37 (WB, 1:1,000, 4793, Cell Signaling), mouse anti-HSP90 (WB, 1:2,000, 610419, BD), mouse anti-FLAG (WB, 1:150,000, F3165, Sigma Aldrich), mouse anti-GAPDH (WB, 1:150,000, H86504M, Meridian Life science), mouse anti-Mitofusin 1 (WB, 1:5,000, ab57602, Abcam), rabbit anti-p38 MAPK (WB, 1:1,000, #9212, Cell Signaling), rabbit anti-PINK1 (WB, 1:2,000, BC100-494, Novus Biologicals), rabbit anti-PINK1 (WB, 1:2,000, IF, 1:100, #6946, Cell Signaling), mouse anti-Parkin (WB, 1:3,000, #4211, Prk8, Cell Signaling), mouse anti-TOM20 (IF, 1:250, sc-17764, Santa Cruz Biotechnology), rabbit anti-TOM20 (WB, 1:20,000, 11802-1-AP, ProteinTech Group), rabbit anti-TOM40 (WB, 1:1,000, 18409-1-AP, ProteinTech Group), mouse anti-Ub (WB, 1:1,000, MAB1510, ubi-1, Millipore), rabbit anti-p-Ser65-Ub (WB, 1:5,000-1:15,000, IF, 1:250, in house [18 (link)]), mouse anti-V5 (WB, 1:5,000, R960-25, Invitrogen), rabbit anti-V5 (WB, 1:5,000, ab9115, Abcam), mouse anti-vinculin (WB, 1:100,000-500,000, V9131, Sigma). Streptavidin HRP (WB, 1:150,0000, Pierce).
Bc100 494
Manufactured by Novus Biologicals
Sourced in United States
The BC100-494 is a laboratory equipment product manufactured by Novus Biologicals. It is designed for use in research and scientific applications. The core function of this product is to provide a specific technical capability, however, a detailed description cannot be provided while maintaining an unbiased and purely factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Ab57602, by Abcam (2 mentions)
Sc 17764, by Santa Cruz Biotechnology (2 mentions)
Ab9115, by Abcam (1 mentions)
V9131, by Merck Group (1 mentions)
Mab1510, by Merck Group (1 mentions)
Ubi 1, by Merck Group (1 mentions)
R960 25, by Thermo Fisher Scientific (1 mentions)
F3165, by Merck Group (1 mentions)
Ab209263, by Abcam (1 mentions)
Topotecan, by Merck Group (1 mentions)
Oligomycin, by Fujifilm (1 mentions)
Hygromycin b, by Fujifilm (1 mentions)
Sc 137243, by Santa Cruz Biotechnology (1 mentions)
Antimycin a, by Fujifilm (1 mentions)
Bafilomycin a1 bvt 0252, by Adipogen (1 mentions)
Ab56889, by Abcam (1 mentions)
Nb100 2220, by Novus Biologicals (1 mentions)
Ab9354, by Merck Group (1 mentions)
Sc 166576, by Santa Cruz Biotechnology (1 mentions)
Bafilomycin a1 bafa1, by Merck Group (1 mentions)
11 protocols using bc100 494
1
Western Blot and Immunofluorescence Protocol
2
Mitochondrial Dynamics and Autophagy Analysis
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The following primary antibodies were used: anti-PINK1 rabbit polyclonal (BC100-494, Novus), anti-PINK1 rabbit monoclonal (6946 S, Cell Signaling Technology), anti-MUL1 rabbit monoclonal (ab209263, abcam), anti-LC3 mouse monoclonal (CTB-LC3-2-IC, Cosmo Bio), anti-ATG14 mouse monoclonal (M184-3, Medical and Biological Laboratories), anti-Multi Ubiquitin mouse monoclonal (D058-3, Medical and Biological Laboratories), anti-Tom20 mouse monoclonal (sc-17764, Santa Cruz Biotechnology), anti-LDHA mouse monoclonal (sc-137243, Santa Cruz Biotechnology), and anti-Actin mouse monoclonal (MAB1501R, Millipore). The following reagents were used: DFP (3-Hydroxy-1,2-dimethyl-4(1 H)-pyridone) (322–65152), oligomycin (O4533), antimycin A (#514-55521), CCCP (carbonyl cyanide 3-chlorophenylhydrazone) (034–16993), and hygromycin B (089–06151), Tetramethylrhodamine Methyl Ester (TMRM) (203–18041) were purchased from FUJIFILM Wako Pure Chemical. The Library of Pharmacologically Active Compounds (LOPAC1280) (LO4200), amsacrine (A9809), camptothecin (C9911), gemcitabine (G6423), and topotecan (T2705) were purchased from Sigma–Aldrich. Bafilomycin A1 (BVT-0252) was purchased from Adipogen Life Sciences.
3
Comprehensive Antibody Validation Protocol
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The following antibodies have been used for western blot (WB) or immunofluorescence (IF): beta III tubulin (#5568, CST, WB: 1/2,000 or AB9354, Millipore, IF: 1/250), FLAG (F3165, Sigma, WB: 1/150,000), GAPDH (H86504M, Meridian Life Sciences, WB: 1/500,000), LC3B (NB100-2220, Novus Biologicals, WB: 1/5,000), Miro1 (H00055288-M01, Novus Biologicals, WB: 1/500), Mitofusin 1 (ab57602, Abcam, WB: 1/5,000), Mitofusin 2 (ab56889, Abcam, WB: 1/5,000), NBR1 (H00004077-M01, Abnova, WB: 1/500, IF: 1/100), NDP52 (12229-1-AP, PTG, WB: 1/1,000, IF: 1/400), OPTN (sc-166576, Santa Cruz, IF: 1/100), OPTN (10837-1-AP, PTG, WB: 1/5,000), p38 (#9212, CST, WB: 1/2,000), p62 (610832, BD Biosciences, WB: 1/2,000, IF:1/500), Parkin (#4211, CST, WB: 1/3,000), PGAM5 (ab12653, Abcam, WB: 1/5,000), PINK1 (#6946, CST, WB: 1/2,000, IF: 1/1,000), PINK1 (BC100-494, Novus Biologicals, WB: 1/2,000), TAX1BP1 (#5105, CST, WB: 1/2,000, IF: 1/400), TBK1 (#3504, CST, WB: 1/1,000), pS172-TBK1 (#5483, CST, WB: 1/1,000), TOM20 rabbit (11802-1-AP, PTG, IF: 1/2,000), TOM20 mouse (sc-17764, Santa Cruz, IF: 1/100), TOM70 (14528-1-AP, PTG, WB: 1/5,000), TRAP1 (#13405, CST, WB: 1/1,000), ubiquitin (#3933, CST, WB: 1/2,000), pS65-Ub (in-house [16 (link), 46 (link)], WB: 1/15,000, IF: 1/250), VDAC1 (ab14734, Abcam, WB: 1/10,000), vinculin (V9131, Sigma, WB: 1/500,000).
4
Antibody Detection for Parkin Pathway Proteins
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Antibodies against Parkin (4211), cleaved caspase 3 (9664), poly(ADP-ribose) polymerase (PARP) (9542), c-Jun (9165), and voltage-dependent anion channel (VDAC) (4866), phospho-SAPK/JNK (Thr183/Tyr185) (9251), horseradish peroxidase (HRP)-linked anti-mouse IgG (7076) were purchased from Cell Signaling Technology (Danvers, MA, United States). Antibodies against p62 (P0067, Sigma-Aldrich, Saint Louis, MO, United States), LC3B (100-2220, Novus Biologicals, City of Centennial, CO, United States), HRP-conjugated β-actin (47778, Santa Cruz Biotechnology, Dallas, TX, United States) and goat anti-rabbit IgG (H+L) secondary antibody (31460, Thermo Fisher Scientific, Carlsbad, CA, United States), PTEN-induced putative kinase 1 (PINK1) (BC100-494, Novus Biologicals) and ubiquitin phosphorylated at S65 residue (p-Ub-S65) (ABS1513-I, EMD Millipore, Burlington, MA, United States) were purchased at the indicated companies. Bafilomycin A1 (Baf.A1, Sigma-Aldrich), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, Sigma-Aldrich), staurosporine (STS, Cell Signaling Technology), and necrostatin-1 (Invitrogen, Carlsbad, CA, United States) were purchased from the indicated companies.
5
Immunohistochemical Analysis of Breast Cancer Proteins
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Immunohistochemical analysis of breast cancer tissues was performed with anti-PINK1 (BC100-494, Novus Biologicals, Centennial, CO, USA), anti-Parkin (sc-32282, Santa Cruz Biotechnology), anti-BRCA1 (MS110, Abcam, Cambridge, UK), and anti-Ki67 (MIB-1, M7240, DAKO, CA, USA) antibodies. Paraffin sections were deparaffinized and rehydrated. Antigen retrieval was performed by autoclaving at 105ºC for 20 min with a retrieval solution pH 9.0 (Nichirei Biosciences Inc., Tokyo) for BRCA1, PINK1 and Parkin, or by autoclaving at 121ºC for 10 min with 1 mM Tris/EDTA pH 9.0 for Ki67. For these analyses, tissues showing atypical ductal hyperplasia were excluded. To quantify BRCA1-positive cells, only nuclear signals were used. For the scoring of PINK1 and Parkin expressions in breast cancer tissues, we compared the signal intensity with normal tissues; a signal was counted as negative only when the signal intensity was lower than the intensity in the normal tissues.
6
Western Blot Analysis of Mitophagy Markers
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Cell lysates were prepared in RIPA buffer plus proteinase inhibitors. Samples containing (20 µg) were separated by 10–15% SDS-PAGE, transferred to nitrocellulose membranes, blocked in 5% nonfat milk for 1h at RT and incubated overnight at 4 °C with the primary antibodies: MFN2 (H-68, Santa Cruz, sc-50331, dilution 1:1000, rabbit); Optineurin (C-2, Santa Cruz, sc-166576, dilution 1:1000, mouse); PINK1 (BC100-494, Novus Biologicals, dilution 1:2000, rabbit); Parkin (PRK8, ab77924, Abcam, dilution 1:2000, mouse); β-Actin (Sigma-Aldrich, A3854, dilution 1:40,000 conjugated to HRP). Secondary antibody incubation was performed for 1 h at RT using anti-mouse (m-IgGκ BP-HRP sc-516102, Santa Cruz, 1:10,000) and anti-rabbit (goat anti-rabbit IgG-HRP sc-2054, Santa Cruz, 1:10,000). Proteins were detected using ECL western blotting substrate (Pierce, Waltham, MA, USA), Clarity and Clarity Max (Bio-Rad, Hercules, CA, USA) and ChemiDoc Imaging System (Bio-Rad).
7
Western Blot Analysis of ATF3 and PINK1
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Protein extraction reagents, MPER or TPER (Thermo Fisher), were used to prepare the protein extracts. Around 20 μg of total lysate was used for electrophoresis in a polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked for 1 hr with Odyssey PBS blocking buffer (Licor). Primary antibodies for ATF3 (sc‐188, Santa Cruz) and PINK1 (BC100‐494, Novus) were incubated overnight. Finally, secondary antibodies (Licor) and Odyssey scanner (Licor) were used. After primary detection, membranes were stripped and re‐probed for β‐actin (sc‐47778, Santa Cruz) as loading control. Quantification was performed using ImageJ software (NIH).
8
Western Blot Analysis of FOXO3, LC3, and S6
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FOXO3 western blots were performed as described previously [44 (link)]. Cells were lysed in Triton lysis buffer (50mM Tris-HCL pH 7.5, 100mM NaCl, 0.5 mM EDTA, 0.4% Triton, 50 mM NaF, 40 mM b-glycerophosphate, 1mM sodium orthovanadate, 1mM PMSF, 0.055 units/ml aprotinin). For LC3 western blots, cells were lysed in 150 mM NaCl, 1.0% Triton, 0.5% Sodium Deoxycholate, 0.05% SDS, 50mM Tris-HCL pH 8 and a cOmplete Mini Protease Inhibitor Cocktail Tablet (Sigma) by shaking at 4°C for 30 minutes. Lysates were centrifuged at max speed for 7 minutes (15 minutes for LC3) and supernatants transferred to new tubes. Protein concentration was quantified on a Qbit using the Qbit Protein Assay Kit (Life Technologies), followed by the addition of Laemmli sample buffer (2% SDS, 10% glycerol, 5% β-meracaptoethanol, 63 mM Tris-HCl pH 6.8, bromophenol blue). All antibodies were diluted in 5% powdered milk or BSA and primary antibody incubations were performed overnight at 4°C. Primary antibodies and concentrations used: Rabbit anti-LC3B 1:1000 (Novus Biologicals NB600-1384), Rabbit anti-FOXO3 1:1000 (CST 75D8), Rabbit anti-FOXO1 1:1000 (Abcam ab12161), Rabbit anti-pS6 1:1000 (CST 4858S), Mouse anti-S6 1:1000 (CST 2317S), Rabbit anti-Pink1 1:1000 (Novus BC100-494) Mouse anti-Actin 1:5000 (Novus Biologicals (NB100-74340).
9
SH-SY5Y Protein Extraction and Western Blot
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SH‐SY5Y cells were harvested with trypsin and lysed on ice in 1% (v/v) Triton X‐100 in PBS supplemented with protease and phosphatase inhibitors or 0.1% (v/v) sodium dodecyl sulphate (SDS), 10 mM Tris pH 7.4, 150 mM NaCl supplemented with DNase and protease/phosphatase inhibitors. Protein lysates (20 μg) were resolved by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to Hybond P (GE Healthcare, Little Chalfont, UK). Blots were probed with antibodies against Nrf2 (ab31163; Abcam, Cambridge, UK), p62 (610833; BD Biosciences, Oxford, UK), prohibitin 1 (2426; Cell Signaling Technology, Beverly, MA, USA), cytochrome oxidase (COX) subunit IV (ab14744; Abcam), LC3B (2775; Cell Signaling Technology), cathepsin D (ab6313; Abcam), TOM20 (FL‐145; Santa Cruz Biotechnology, Santa Cruz, CA, USA), TFEB (ab2636; Abcam), DDK (clone 4C5; Origene), lamin A (ab26300; Abcam), PINK1 (BC100‐494; Novus Biologicals, Cambridge, UK), PGC‐1α (NBP1‐04676; Novus Biologicals) or β‐actin (ab82618; Abcam). Bands were detected with respective horse radish peroxidase‐linked secondary antibodies (Dako, Carpinteria, CA, USA) and enhanced chemiluminescence (Pierce). Density of bands was determined using ImageJ software (NIH, Bethesda, MD, USA) or Image Lab software 5.1 (Bio‐Rad Laboratories, Hercules, CA, USA).
10
Antibody Generation for PINK1 Protein
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Rabbit polyclonal antibody B2 was generated using human PINK1 sequences (175–250 aa) by Boster Biological Technology (China). The same human PINK1 sequences were used to produce a rabbit antibody (BC100–494, Novus Biologicals, USA), widely used to recognize PINK1 in cultured cells. The human PINK1 sequences were also used as antigens to generate the mouse monoclonal antibody E7B6 by Nanjing Detai Bioengineering (China). Additionally, a synthetic peptide containing the pig PINK1 protein sequences (residues 484–504) was used as an antigen to produce a rabbit polyclonal antibody (anti-pig PINK1) by Boster Biological Technology (China). Other antibodies used in the study are listed in Supplementary Table S1.
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