M per r mammalian protein extraction reagent

The cells were gathered and broken down in M-PER (R) Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, USA) while being kept on ice for a duration of 30 min. The cell lysates underwent resolution through SDS-PAGE and were subsequently transferred to PVDF membranes (Cytiva, USA). The PVDF filters were treated with QuickBlock™ Western blocking solution (Beyotime, China) for 1 h while gently shaking. Subsequently, the filters were incubated with the specified antibodies at 4 °C overnight. Goat secondary antibodies conjugated with HRP (1:1000, Beyotime, China) were employed. After being washed with TBST, the membranes underwent a thorough cleansing, followed by incubation of the secondary antibody at room temperature for 2 h. To examine the protein bands, we employed a chemiluminescent western blot detection method.

Certain number of cells were collected and total cellular proteins were extracted in M-per(R) Mammalian Protein Extraction Reagent (Thermo Scientific). Cellular proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and transferred to poly-vinylidene fluoride membranes (Millipore; Bedford, MA) for immunoblotting. After blocking with 5% skim milk (BD Biosciences), the membrane was probed with the primary antibodies rabbit monoclonal to GPCR RDC1 (abcam, Cambridge, MA.ab138509,1:1000 diluted) overnight at 4 °C. After washing, membranes were labeled with the horseradish peroxidase conjugated goat anti-rabbit secondary antibodies (R&D Systems,1:4000) for 1 h at room temperature. The immunoblots were colored by using an ECL Kit (Millipore). Anti-actin was used for loading control (Sigma-Aldrich). Blots were detected and imaged by Image Lab (Bio-Rad).