Ab8226

After extraction, protein concentrations were tested using a BCA protein assay kit (Pierce Co, Rockford, IL, USA). Equal amounts of protein were run in 10% SDS-PAGE gel and transferred to a polyvinylidene difluoride (PVDF) membrane and then incubated with the primary antibodies at 4 °C overnight. The antibodies included anti-ASPM rabbit polyclonal antibody (1:1000, A02584-1, Boster, China), Mouse monoclonal [mAbcam 8226] to beta Actin - Loading Control (1:1000, AB8226, abcam, USA). After incubation with secondary antibody (1:2000; Santa Cruz), the signals were visualized and analyzed through image acquisition and analysis software (Bio-Rad, Hercules, CA, USA).

The lysis buffer was used to extract the total protein from PTC tissues or cells. After the protein concentration was determined by the BCA protein assay kit (Abcam), 50 µg of protein was separated by a 10% SDS-PAGE and then transferred to the PVDF membrane. The PVDF membrane was blocked with 5% skim milk at 37 °C for 2 h and then incubated overnight at 4 °C with the following primary antibodies: Anti-Succinyllysine (1: 500, PTM-401, PTMBIO, Hangzhou, China), SIRT7 (1: 200, ab259968, Abcam), LATS1 (1: 200, ab243656, Abcam), and β-actin (1: 200, ab8226, Abcam). The membrane was washed 3 times with TBST, 10 min each time. Then, the secondary antibody (1: 1000, AF008, Novus, Shanghai, China) was incubated for 2 h at room temperature, and the membrane was washed by TBST for 3 times, 10 min each time. Finally, band signals were visualized using an enhanced chemiluminescence solution (Thermo Fisher Scientific, Monmouth Junction, NJ, USA) and exposed in a ChemiDocTM XRSC system (Bio-Rad).

The skin tissues were milled, and then, protein extraction and determination were performed with the Total Protein Extraction Kit and BCA Protein Concentration Kit. Prepare 8–12% SDS-PAEG gel electrophoresis to separate the total protein and then transfer to PVDF membranes. After the membrane transfer was completed, the protein was blocked with T-TBS (containing 5% skimmed milk powder) at room temperature for 1 h and rinsed with T-TBS three times for 5 min each. After incubation overnight at 4 °C with primary antibodies of Integrin (1:5000, Abcam, ab179475), EGFR (1:1000, ab52894, Abcam), LATS1 (1:1000, 3477S, Cell Signaling Technology), YAP (1:1000, 14074S, Cell Signaling Technology), p-YAP (1:1000, 13008S, Cell Signaling Technology), GPCR (1:2000, ab183127, Abcam), and β-actin (1:1000, ab8226, Abcam), the membranes were washed and incubated with goat anti-rabbit IgG (H + L) (1:10,000, 31210S, Thermo Pierce) as the secondary antibody for 1 h at room temperature, except for β-actin, which was incubated with goat anti-rat IgG (H + L) (1:10,000, 31160S, Thermo Pierce) as the secondary antibody.
Use SuperSignal® West Dura Extended Duration Substrate, prepare about 1 mL of ECL working solution following the instructions, incubate the transfer membrane at room temperature for 1 min, then remove the excess ECL reagent, seal it with plastic wrap, and place the X-ray film in the cassette for exposure for 5–10 min, developing and fixing.