Ripi lysis buffer

Briefly, total protein from the P6 human disc NP cells was extracted using RIPI lysis buffer (Beyotime, China). Then, protein supernatant samples were separated by SDS/PAGE and transferred on to the PVDF membranes. Subsequently, the PVDF membranes were incubated with primary antibodies (β-actin: Abcam, ab8226; p16: Abcam, ab108349; p53: Abcam, ab1101; Akt: Cell Signaling Technology, #4685; p-Akt: Cell Signaling Technology, #9271) at 4°C overnight and second antibodies at 37°C for 2 h. Finally, protein bands were visualized using a BeyoECL Plus Kit (Beyotime, China) and analyzed using the ImageJ software.

Cells were lysed in RIPI Lysis Buffer (Beyotime) supplemented with protease inhibitors and phosphatase inhibitors (Bimake). Protein concentrations were measured with the BCA reagent (Beyotime) by using a Beckman Coulter DU‐800 spectrophotometer. Equal amounts of protein were resolved by SDS‐PAGE and immunoblotted with Piezo1 Monoclonal Antibody (Invitrogen, MA5‐32876) at a dilution of 1:1000. The immunoblots were detected using an Odyssey Fc Imaging System (LI‐COR).