Frozen muscle samples were homogenized in Western blot lysis buffer (Beyotime, China) and centrifuged at 12,000×g for 5 min at 4°C. The total protein concentration of the supernatant was determined (BioRad, Hercules, CA, USA). Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred to polyvinylidene difluoride membranes, blocked with 5% milk powder, and incubated with primary antibodies against TGF-β1 (Santa Cruz Biotechnology), Smad3 (Santa Cruz Biotechnology), and HDAC4 (Abcam, Cambridge, UK) at 4°C overnight and. with horseradish peroxidase-conjugated anti-rat IgG secondary antibody (60 min, room temperature). Signals were detected using a chemiluminescence system (Pierce Biotechnology, Rockford, IL, USA) and quantified using an image analysis system (Bio-Rad).
Western blot lysis buffer
Manufactured by Beyotime
Sourced in China
Western blot lysis buffer is a solution used to extract and solubilize cellular proteins for Western blot analysis. It contains a combination of detergents, salts, and buffering agents that help lyse cells and denature proteins, allowing for their efficient separation and detection.
Automatically generated - may contain errors
Lab products found in correlation
Image analysis system, by Bio-Rad (1 mentions)
Chemiluminescence system, by Thermo Fisher Scientific (1 mentions)
Smad3, by Santa Cruz Biotechnology (1 mentions)
Hdac4, by Abcam (1 mentions)
Tgf β1, by Santa Cruz Biotechnology (1 mentions)
Protein a g agarose bead, by Beyotime (1 mentions)
Gapdh, by Merck Group (1 mentions)
α actinin, by Merck Group (1 mentions)
Rictor, by Abcam (1 mentions)
Hsp90, by Abcam (1 mentions)
Connexin 43, by Abcam (1 mentions)
Rictor, by Santa Cruz Biotechnology (1 mentions)
Mitochondrial protein extraction kit, by Beyotime (1 mentions)
Vdac1, by Santa Cruz Biotechnology (1 mentions)
Akt1 2 3, by Santa Cruz Biotechnology (1 mentions)
Acetyl lysine, by Abcam (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Pierce ecl western blotting substrate kit, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Bca kit, by Beyotime (1 mentions)
5 protocols using western blot lysis buffer
1
Western Blot Analysis of TGF-β1, Smad3, and HDAC4
2
Mitochondrial Protein Extraction and Co-Immunoprecipitation
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Cell lysates were prepared in Western blot lysis buffer (Beyotime, Shanghai China). Mitochondrial/cytoplasmic proteins were obtained from samples with a mitochondrial protein extraction kit (Beyotime). Samples were coimmunoprecipitated using Protein A + G agarose beads (Beyotime) according to the manufacturer’s instructions. Western blotting was performed as previously reported [26 (link), 27 (link)]. Antibodies against thefollowing proteins were used: GAPDH, α-actinin (Sigma Aldrich), Akt1/2/3 (Ser 473), Akt1/2/3, Rictor, VDAC1 (Santa Cruz, TX, USA), cytochrome c, Oct4, mTOR, p-mTOR (Ser2481), HDAC6, TOM20 (Cell Signalling Technology, MA, USA), c-TNT, Connexin 43, Rictor, Hsp90, SIN1, G protein beta subunit like, and acetyl-lysine (Abcam, MA, USA). The membranes were incubated with horseradish peroxidase (HRP)-conjugated antibodies (Lianke, Hangzhou, China). All data analyses were carried out by using ImageJ software.
3
Western Blot Analysis of Chondrocyte Markers
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We harvested and lysed cells in western blot lysis buffer (Beyotime Biotechnology, Haimen, China). Then, we measured total protein concentrations with BCA kit (Beyotime Biotechnology). Equal amounts of protein were loaded and separated on a SDS-PAGE gel and transferred to a polyvinylidene fluoride membrane (Millipore Billerica, MA, USA). Then, the membranes were incubated with the corresponding primary antibodies (rabbit anti-human GAPDH, Abcam, ab181602; rabbit anti-human Sox9, Abcam, ab185230; rabbit anti-human COL2, Abcam, ab34712; rabbit anti-human AGN, Abcam, ab36861; rabbit anti-human Runx2, Cell Signaling, #12556; mouse anti-human COL1, Abcam, ab34710; rabbit anti-human ALP, Abcam, ab83259; and mouse anti-human O-GlcNAcyaltion antibody (RL2), Thermo, MA1-027) respectively, overnight at 4 °C, washed three times in PBS, and incubated with the corresponding secondary antibody (anti-rabbit IgG-HRP-linked antibody, #7074, Cell Signaling; anti-mouse IgG-HRP-linked antibody, #7076, Cell Signaling) for 1 h. The membranes were washed three times in PBS and subjected to western blotting using a Pierce ECL western blotting substrate kit (Thermo Scientific, MA, USA). Immunoreactive proteins were measured with chemiluminescent detection.
4
Western Blot Analysis of UPR Markers
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Cells were washed with cold PBS and lysed with western blot lysis buffer (Beyotime, China), and the lysates were centrifuged at 13 400 g for 5 min at 4°C. Protein concentrations were quantified using a BCA Assay Kit (Beyotime, China). Proteins were separated by 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membrane was incubated overnight at 4°C with antibodies against GAPDH (Wanleibio, China), BIP, PERK, IRE1α, phospho IRE1α, EIF2α and phospho EIF2α (all from Cell Signaling Technology, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (ZSGB-BIO, China) was incubated with the membrane at 37°C for 1 h. Proteins were detected by chemiluminescence using SuperSignal West Pico (Thermo Fisher, USA) and imaged on a ChemiDoc MP imaging system (Bio-Rad, USA).
5
Western Blot Analysis of NF-κB Pathway
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Cells were seeded in 60-mm dishes at 2 × 106 cells/dish and grown until confluent. After specific treatment, cells were collected and lysed in 500 μL of Western blot lysis buffer (Beyotime, Shanghai, China) supplemented with the proteinase inhibitor phenylmethylsulfonyl fluoride (Sigma-Aldrich). The protein concentration was determined by the bicinchoninic acid assay (BIOSHARP, Hefei, China), and equivalent amounts of protein were separated on sodium dodecyl sulfate polyacrylamide gel electrophoresis (5%–12% polyacrylamide) and then transferred to a polyyinylidene difluoride membrane. The membrane was blocked with 5% bovine serum albumin (BSA) in 0.1 M Tris-buffer saline (pH 7.5) containing 0.05% Tween-20 (TBST) for at least 1 h, and then probed overnight with appropriate primary antibodies against as follows: IκB-α, p-p65, p65 or β-actin (1:2000 in 5% BSA in TBST; Cell Signaling Technology, Danvers, MA, USA). They were then incubated with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies for 2 h at 37 °C. Blots were finally visualized with ECL reagents (Bio-Rad, Hercules, CA, USA) via chemiluminescence. To compare reaction intensities, average band densities were determined using Quantity One software (Bio-Rad).
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