P taz

The total protein extraction kit (Teyebio, Shanghai, China) was used to lyse and extract tissue proteins and cellular proteins. The specific experimental operation can refer to the previous research [17 (link)]. The protein concentration was subsequently determined using the BCA method (Teyebio, Shanghai, China). After the denatured protein was separated by gel running, the resultant was blotted onto a polyvinylidene fluoride membrane. The entire transfer system was placed in an ice-water mixture, and the membrane was transferred for about an hour under the conditions of 100 V, 400 mA. This was followed by overnight incubation with 5% nonfat dry milk in blocking solution. Use the desired antibody as the primary antibody to incubate the blocked PVDF membrane according to the instructions, and add an appropriate amount of secondary antibody and incubate with shaking at room temperature. A development kit (Teyebio, Shanghai, China) visualized the bands. The antibodies used are as follows: CyclinD1, p21, MMP9, snail, YAP1, p-YAP, TAZ, p-TAZ, GAPDH antibodies (1 : 2000, Abcam), and HRP labeled IgG antibody (1 : 10000, Cell Signaling Technology).

Total protein extraction from incorporated cells was conducted using RIPA lysis buffer (WB-0071, Dingguo Changsheng Biotech, Beijing), and protein quantification utilized the BCA protein quantitative kit (BCA01, Dingguo Changsheng Biotech, Beijing). Subsequently, we loaded (20 μg) on 10% SDS-PAGE gel for protein separation, before transfer to PVDF membranes, which were then treated with specific primary antibodies as follows: GALNT2 (Abcam, Shanghai, Ab140637, 1:2000), LATS1 (Abcam, Shanghai, Ab243656, 1:1000), LATS2 (Abcam, Shanghai, Ab243657, 1:1000), p-LATS2 (Proteintech, 28998-1-AP, 1:2000), YAP (Abcam, Shanghai, Ab205270, 1:1000), p-YAP (Abcam, Shanghai, Ab76252, 1:5000), TAZ (Abcam, Shanghai, Ab242313, 1:1000), p-TAZ (Abcam, Shanghai, Ab277791, 1:1000), β-actin (Proteintech, 81115-1-RR, 1:2000), and GAPDH (PROTEINTECH; 60004-1, 1:5000). Protein visualization utilized the ECL reagent (ECL-0111, Dingguo Changsheng Biotech, Beijing) and protein quantification was performed using Image J, with GAPDH or β-actin as the control.