Whole-cell lysates were obtained and denatured in a 5×SDS loading buffer at 100°C for 10 minutes with the guidance as described previously.17 (link) Proteins in the samples were separated by SDS-PAGE (Biorad, 1611210), semi-dry electrophoretic transferred to PVDF membrane (Biorad, 1704156), and analyzed using the specified antibodies and an ECL detection system (Thermo Fisher Scientific) with Hsp90 used as a loading control. The primary antibodies were incubated as follows: anti-Prx1 (1:2000, Abcam), anti-p53 (1:1000, CST), anti-p21 (1:2000, Abcam), anti-CDK2 (1:1000, Abcam), anti-CDK4 (1:2000, Abcam), anti-CDK6 (1:500,CST), anti-PHB2 (1:10000, Abcam), anti-LC3B (1:1000, Abcam) and anti-Hsp90 (1:2000, Santa).
Protocol detail
Western Blot Analysis of Cell Signaling Proteins
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Antibodies
Buffer
Cdk2
Cdk6
Cell
Electrophoretic
Hsp90
Membrane
Phb2
Proteins link
Prx1
Pvdf
Sds page
Corresponding Organization : Capital Medical University
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Variable analysis
independent variables
- None explicitly mentioned
- Protein expression levels of Prx1, p53, p21, CDK2, CDK4, CDK6, PHB2, and LC3B
- Hsp90 as a loading control
- Positive control: None mentioned
- Negative control: None mentioned
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