Total proteins were collected using RIPA buffer (Thermo Scientific, CA, USA) and then quantified for protein quantification by using Pierce™ BCA Protein Quantification Kit (Thermo Scientific) [15 (link)]. Protein (20 μg) was used for electrophoresis loading SDS-PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk and incubated with primary antibodies, including anti-KIAA1429 (Cell Signaling Technology, 1:1000, #88,358), anti-ROCK2 (Cell Signaling Technology, 1:1000, #47,012), and beta-actin (CWBio, Beijing, China). After incubation by primary antibodies or their corresponding secondary antibodies, blots were developed using SuperSignal West Dura Persistence Substrate (Thermo Scientific).
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Protein Quantification and Western Blotting
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Corresponding Organization : Tianjin Hospital
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Variable analysis
independent variables
- None explicitly mentioned
- Protein levels of KIAA1429 and ROCK2
- Total protein quantification using Pierce™ BCA Protein Quantification Kit
- Protein loading (20 μg) for electrophoresis
- Blocking with 5% skimmed milk
- Primary antibodies used: anti-KIAA1429, anti-ROCK2, and beta-actin
- Secondary antibody incubation
- Blot development using SuperSignal West Dura Persistence Substrate
- Positive control: beta-actin
- Negative control: Not explicitly mentioned
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