Beta actin polyclonal antibody

Colon tissues were homogenized and lysed on ice for 30 min in RIPA lysis buffer (Solarbio). Protein concentrations were determined by using a bicinchoninic acid protein assay kit (ABP Biosciences). Protein extracts were mixed with 5× SDS-PAGE Sample Loading Buffer (Beyotime), boiled at 95°C for 10 min, loaded into the 4–8% SDS-PAGE gel for electrophoresis, and transferred to the polyvinylidene difluoride membrane (Millipore), successively. After blocking with 5% non-fat milk, the membranes were incubated with Piezo1 Polyclonal antibody (1:400, 15939-1-AP; proteintech) and Beta Actin Polyclonal antibody (1:5,000, 20536-1-AP; proteintech) overnight at 4°C. Next, the membranes were washed and incubated with HRP-conjugated anti-rabbit secondary antibody (1:5,000, ZB-2301; ZSGB-BIO) for 1 h at room temperature. The membranes were visualized in an enhanced chemiluminescence (Millipore). The chemiluminescent density of protein bands was quantified using Quantity One (Bio-Rad).

The protein extraction was conducted when cells were grown to a density of 100% confluence. In brief, cells were added with RIPA lysis buffer and then shocked on the ice for 2h. After frozen at −80 °C overnight, the mixture was centrifuged 30 min at 4 °C and the protein was drawn from the supernatant. The protein mixed with 1× loading buffer was loaded onto 10% SDS-PAGE gel and then transferred to a nitrocellulose membrane for further exposure. The primary antibodies were purchased from Proteintech (GSK3B polyclonal antibody, phospho-Gsk3b polyclonal antibody, betacatenin polyclonal antibody, C-MYC polyclonal antibody, beta actin polyclonal antibody).