Cell extracts were prepared using RIPA buffer (20mM Tris, pH 7.4, 137 mM NaCl, 1% NP-40, 0.25% Deoxycholate, 0.1% SDS, 1mM EDTA and 1% protease inhibitor cocktail). Protein concentration was determined by BCA kit (Pierce; Rockford, IL) and 100 µg protein was analyzed by Western blotting as described previously69 (link). Anti-p53 (sc-98, Santa Cruz) was used at 1:1000 (2 µg/mL) dilution and anti-Bcl-2 (sc-492, Santa Cruz) and anti-β-actin (Sigma) were used at 1:1000 dilutions. SPDEF antibodies (gift from Dr. Jeffery Whitsett, Children’s Hospital, University of Cincinnati, OH) were used at 1:5000 dilution. Proteins were detected using ECL and visualized by chemiluminescence (Perkin Elmer, Waltham, MA) using the FujiFilm Image Reader LAS-4000 (Valhalla, NY). The uncropped scans of the most important Western blots are included as a supplementary figure in the Supplementary Information.
Anti bcl 2 sc 492
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Anti-Bcl-2 (sc-492) is a laboratory product for research purposes. It is an antibody that targets the Bcl-2 protein, which is involved in the regulation of apoptosis, or programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (3 mentions)
Anti bax sc 493, by Santa Cruz Biotechnology (3 mentions)
Image reader las 4000, by Fujifilm (2 mentions)
Anti p53 sc 98, by Santa Cruz Biotechnology (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ab3623, by Merck Group (1 mentions)
Anti t foxo3a, by Cell Signaling Technology (1 mentions)
Anti p foxo3a, by Cell Signaling Technology (1 mentions)
Anti active caspase 3, by Merck Group (1 mentions)
Anti t akt, by Cell Signaling Technology (1 mentions)
Anti p akt ser473, by Cell Signaling Technology (1 mentions)
Goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Nacalai Tesque (1 mentions)
Anti bcl xs l, by BioLegend (1 mentions)
Anti β actin, by BioLegend (1 mentions)
Anti caspase 8, by Medical & Biological Laboratories (1 mentions)
Goat anti mouse alkaline phosphatase conjugated secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Anti caspase 3, by Cell Signaling Technology (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000 system, by Fujifilm (1 mentions)
7 protocols using anti bcl 2 sc 492
1
Western Blot Analysis of Protein Expression
2
Molecular Mechanisms of Kidney Injury
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The following primary antibodies were used for immunoblot analysis or immunohistochemical staining: anti-KIM-1 (ab56015; Abcam, Cambridge, UK), anti-8-OHdG (MOG-100 P; JaICA, Shizuoka, Japan), anti-4-HHE (MHH-030n; JaICA), anti-MnSOD (ab16953; Abcam), anti-PI3K (610045; BD Transduction Laboratories, San Jose, CA, USA), anti-p-AKT (Ser473) (9271 S; Cell Signaling Technology, Danvers, MA, USA), anti-t-AKT (9272 S; Cell Signaling Technology), anti-p-FoxO3a (9466 S; Cell Signaling Technology), anti-t-FoxO3a (2497 S; Cell Signaling Technology), anti-β-actin (A5441; Sigma-Aldrich), anti-COX-IV (A301-899 A; Bethyl Laboratories, Montgomery, TX, USA), anti-Bcl-2 (sc-492; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Bax (DB005; Delta Biolabs, Gilroy, CA, USA), anti-active caspase-9 (9505 S; Cell Signaling Technology), and anti-active caspase-3 (AB3623; Millipore Corporation, St. Charles, MO, USA).
3
Western Blot Analysis of Apoptosis Regulators
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Cells were lysed with the M-PER mammalian protein extraction reagent (Thermo Scientific, Rockford, IL) containing a protease inhibitor cocktail (Nacalai Tesque). Equal amounts of protein were resolved on 4–12% gradient or 12% SDS-PAGE gels, followed by transfer to polyvinylidene fluoride membranes. After blocking membranes, blots were incubated with the indicated primary antibodies: anti-Bcl-2 (sc-492; Santa cruz biotechnology [SCB], Dallas, Texas, USA, anti-Bcl-XS/L (#633901; BioLegend, San Diego, CA, USA ), anti-Mcl-1 (sc-819; SCB ), anti-caspase-3 (9668; Cell Signaling Technology [CST], Danvers, MA, USA ), anti-caspase-8 (M032-3; Medical and Biological Laboratories, Nagoya, Japan), anti-caspase-9 (9508; CST), anti-β-actin (BioLegend ) or anti-α-tubulin (SCB). After washing, room temperature incubation of membranes for 30 min with either goat anti-rabbit or goat anti-mouse alkaline phosphatase-conjugated secondary antibodies (Invitrogen) were used to detect the primary antibodies. Protein bands were visualized using an ImageQuant LAS-4000 system (FujiFilm, Tokyo, Japan).
4
Western Blot Analysis of Protein Expression
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Cell extracts were prepared using RIPA buffer (20mM Tris, pH 7.4, 137 mM NaCl, 1% NP-40, 0.25% Deoxycholate, 0.1% SDS, 1mM EDTA and 1% protease inhibitor cocktail). Protein concentration was determined by BCA kit (Pierce; Rockford, IL) and 100 µg protein was analyzed by Western blotting as described previously69 (link). Anti-p53 (sc-98, Santa Cruz) was used at 1:1000 (2 µg/mL) dilution and anti-Bcl-2 (sc-492, Santa Cruz) and anti-β-actin (Sigma) were used at 1:1000 dilutions. SPDEF antibodies (gift from Dr. Jeffery Whitsett, Children’s Hospital, University of Cincinnati, OH) were used at 1:5000 dilution. Proteins were detected using ECL and visualized by chemiluminescence (Perkin Elmer, Waltham, MA) using the FujiFilm Image Reader LAS-4000 (Valhalla, NY). The uncropped scans of the most important Western blots are included as a supplementary figure in the Supplementary Information.
5
Western Blot Analysis of Cellular Proteins
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Cells were lysed with radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China). After the protein concentration was determined with BCA protein assay kit (Thermo Fisher Scientific), equal protein aliquots (25 μg per lane) were resolved on SDS gel electrophoresis and Western blotting assay was performed with standard protocol. The expression of target proteins was detected by using enhanced chemiluminescence system (Bio-Rad, Richmond, CA, USA) and quantified by densitometry with Image J software (http://rsb.info.nih.gov/ij/ , Bethesda, MD, USA). Protein expression was normalized by the loading control (GAPDH) expression. Antibodies against FAM46C (ab169699), CDK1 (ab32384), XIAP (ab2541) and Ras (ab52939) were purchased from Abcam (Cambridge, MA, USA). Anti-Bcl-2 (sc-492) and anti-Bax (sc-493) were from Santa Cruz Biotech. (Santa Cruz, CA, USA). Antibodies against Cyclin B1 (#4135), PCNA (#13110), p-MEK1/2 (#8211), MEK1/2 (#9122), p-ERK1/2 (#4376), ERK (#4695) and GAPDH (#5174) were purchased from Cell Signaling (Danvers, MA, USA).
6
Hyperoside Regulation of NF-κB Pathway
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Hyperoside (C21H20O12, purity ≥ 99%, relative molecular mass = 464.38) was from Despite Biotech (Chengdu, China) (Figure 7 ). Primary antibodies for β-actin (#3700), anti-p-NF-κB p65 (#3033), anti-NF-κB p65 (#8242), anti-p-IκBα (#2859), and anti-IκBα (#9242) were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-cleaved caspase-3 (#ab184787) and anti-cleaved PARP (#ab32064) were obtained from Abcam (Cambridge, UK). Anti-Bax (#sc-493) and anti-Bcl-2 (#sc-492) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). In addition, we bought the N-acetyl-cysteine (NAC) from Sigma-Aldrich Chemical (Shanghai, China). IκB-α inhibitor BAY11-7082 and annexin V-FITC Apoptosis Detection Kit were purchased from the Beyotime Institute Biotechnology (Shanghai, China).
7
Western Blot Antibody Profiling
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The following antibodies and reagents were used for the Western blotting experiments. Anti-LC3 (5F10, Nanotools), anti-S6 (2217, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-S6 (4858, Cell Signaling Technology), anti-cleaved caspase 3 (9664, Cell Signaling Technology), anti-SQSTM1/p62 (P0067, Sigma-Aldrich, Saint-Louis, MO, USA), anti-Bcl-2 (sc-492, Santa Cruz Biotechnology, Dallas, TX, USA), and anti-Bax (sc-493, Santa Cruz Biotechnology). Anti-GAPDH (G8715, Sigma-Aldrich) served as an internal control. Secondary antibodies are HRP-linked anti-mouse IgG (7076, Cell Signaling Technology) and HRP-linked anti-rabbit IgG (1706515, Bio-Rad Laboratories, Hercules, CA, USA).
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