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3 protocols using anti bcl2 and anti bax

1

Protein Expression Analysis in Cardiac Cells

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H9c2 cells and cardiac tissue were lysed with RIPA including PMSF (Phenylmethanesulfonyl fluoride, Beyotime, Shanghai, China). The concentration of protein was checked by BCA Protein Assay Kit (Beyotime, Shanghai, China). Subsequently, the protein was separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The primary antibodies included: anti-GRP78 (1:1000, proteintech, Wuhan, China), anti-CHOP and anti-F4/80 (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-Bcl2 and anti-Bax (1:500, Santa Cruz Biotechnology, Dallas, TX, USA), anti-β actin (1:1000, BOSTER, Wuhan, China), anti-GAPDH (1:1000, Beyotime, Shanghai, China). Secondary antibodies were: HRP-Conjugated AffiniPure Goat Anti-Rabbit IgG and HRP-Conjugated AffiniPure Goat Anti-Mouse IgG (1:2000, ZSGB-BIO, Beijing, China). Immunoreactive proteins were visualized using ECL Western blot detection reagents (Cell Signaling Technology, USA).
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2

Western Blot Analysis of Cell Signaling

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Tissue samples and cells were lysed in ice-cold radioimmunoprecipitation assay buffer buffer (Beyotime, Shanghai, China) with fresh-added protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA). Total cell lysates with equal amount of protein for each sample were loaded to sodium dodecyl sulfate-polyacrylamide gel and then transferred to nitrocellulose membranes (Millipore, Bradford, MA, USA). The membranes were incubated with 5% non-fat milk in Tris buffer containing Tween-20 (TBST) for 1 h at room temperature to block non-specific binding. Subsequently, the membranes were probed with primary antibodies (anti-STK39 and anti-c-Myc [Abcam, Cambridge, MA, USA]; anti-Bcl-2 and anti-Bax [Santa Cruz Biotech, Santa Cruz, CA, USA]; anti-p53, anti-p-p38, anti-p38, and anti-GAPDH [Cell Signaling Technology, Danvers, MA, USA]) following the manufacturers’ instructions. After washing three times with TBST, the corresponding horseradish peroxidase-conjugated secondary antibodies (Beyotime) were added. After incubation for 1 h at room temperature and after washing three times with TBST, signals were detected with enhanced chemiluminescence system (Bio-Rad, Richmond, CA, USA). The band density was quantified using ImageJ (http://rsb.info. nih.gov/ij/, Bethesda, MD, USA).
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3

Western Blot Analysis of Apoptosis Markers

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Samples of protein fraction containing equal amounts of protein per lane (40 μg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For Western blot analysis, separated proteins were transferred from gel to a nitrocellulose membrane. The quality of the transfer was controlled by Ponceau S staining of nitrocellulose membranes after the transfer and protein loading by using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a housekeeper. Specific anti-Akt kinase, anti-Bcl-2 and anti-BAX (from Santa Cruz Biotechnology) and antiphospho (P)-Akt kinase (from Cell Signaling Technology) antibodies were used for the primary immunodetection.
Peroxidase-labelled anti-rabbit immunoglobulin (Cell Signaling Technology) was used as the secondary antibody. Bound antibodies were detected by the enhanced chemiluminiscence detection method using Carestream PC program. Protein levels of Akt, P-Akt and BAX were normalized to GAPDH. PI3K/Akt activity was expressed as a ratio of P-Akt and Akt. ColorBurst marker (Sigma Aldrich) was used for quantitative molecular mass determination of transferred proteins.
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