Western Blotting Protein Expression Analysis

Western blotting was used to verify the expressed proteins as previously described.18 (link) Briefly, gastrocnemius and C2C12 cells were lysed with RIPA buffer (Solarbio, Beijing, China), and protein was quantified by using a BCA protein assay kit (Beyotime, Shanghai, China). Forty micrograms of the samples were separated on 10–12% SDS‐polyacrylamide gels and electrophoretically transferred onto PVDF membranes (Millipore, USA). After blocking with 5% fat-free milk in Tris‐buffered saline (TBS) for 1 hour, the membranes were incubated with primary antibodies against HDAC2, P53, P21, IKK, NF-κBp65 (all 1:1000, Cell Signaling Technology, USA), MURF1, MAFbx, and SMP30 (all 1:1000, Abcam, UK) overnight at 4 °C overnight, followed by incubation with fluorescent secondary antibodies (1:1000, Cell Signaling Technology, USA) for 1 hour. Quantification of signal intensities was quantified using ImageJ software.

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Li C., Deng Z., Zheng G., Xie T., Wei X., Huo Z, & Bai J. (2022). Resveratrol Prevents Skeletal Muscle Atrophy and Senescence via Regulation of Histone Deacetylase 2 in Cigarette Smoke-Induced Mice with Emphysema. Journal of Inflammation Research, 15, 5425-5437.

Publication 2022

RIPA buffer BCA protein assay kit PVDF membranes HDAC2 NF-κBp65 MURF1 MAFbx SMP30 fluorescent secondary antibodies

Antibodies Assay Buffer Cells Fluorescent antibodies Free Gastrocnemius Membranes Milk Murf1 Polyacrylamide gels Protein Pvdf Ripa Saline

Corresponding Organization :

Other organizations : Hunan Provincial People's Hospital, Zhuzhou Central Hospital, Guangxi Medical University, First Affiliated Hospital of GuangXi Medical University

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Variable analysis

independent variables

Not explicitly mentioned

dependent variables

Protein expression levels of HDAC2, P53, P21, IKK, NF-κBp65, MURF1, MAFbx, and SMP30

control variables

Gastrocnemius and C2C12 cells were lysed with RIPA buffer (Solarbio, Beijing, China)
Protein was quantified by using a BCA protein assay kit (Beyotime, Shanghai, China)
40 micrograms of the samples were separated on 10–12% SDS‐polyacrylamide gels
Proteins were electrophoretically transferred onto PVDF membranes (Millipore, USA)
Membranes were blocked with 5% fat-free milk in Tris‐buffered saline (TBS) for 1 hour
Primary antibodies were used at 1:1000 dilution
Fluorescent secondary antibodies were used at 1:1000 dilution
Signal intensities were quantified using ImageJ software

controls

Not explicitly mentioned
Not explicitly mentioned

Annotations

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