Anti mouse secondary antibodies

Cells were seeded on a 4-chamber 35 mm-glass bottom dish (in vitro Scientific). Where indicated, cells were treated with the indicated treatments, then fixed with 3.7% paraformaldehyde for 15 min and blocked with 1% BSA in PBS for 1 h. Samples were labeled with the indicated primary antibodies overnight at 4°C in the presence of 0.01% saponin and 1% BSA. Samples were washed 3 times and subsequently incubated with the appropriate combinations of Alexa Fluor (488 or 594)-conjugated anti-rabbit, anti-rat, or anti-mouse secondary antibodies (Thermo Fisher scientific). Samples were analyzed with a Zeiss LSM 710 laser scanning confocal microscope (LSCM) attached to a Zeiss Observer Z1 microscope, using a 63 × oil Plan Apo, 1.4 NA objective. Images were collected using ZEN-LSM software keeping the laser power and gain constant during all acquisitions for comparative analysis of wild type and knockout cells or treated with vehicle or various treatments. Images were then processed using ImageJ (National Institutes of Health, Bethesda, MD) and Photoshop CS4 (Adobe). The fluorescence intensities were quantified using the ImageJ software.

Collected cell samples were lysed by RIPA lysis buffer (Beyotime, China) on ice for 30 min, and then lysis mixture was centrifugated at 12 000 rpm for 10 min. The supernatant was harvested, and the concentration was assessed using a BCA kit. Samples were diluted to a same concentration and added 5× SDS loading buffer according to the volume of the lysate, and samples were boiled at 100 °C for 5 min. Then, specimens were separated on 8–12% SDS‐PAGE and transferred onto PVDF membranes. The PVDF membranes were blocked with 5% milk/TBST at room temperature for 1 h. Primary antibodies against p‐RSK2 (T577) (1:500; sc‐374664, Santa Cruz), RSK2 (1:500; sc‐9986, Santa Cruz), GAPDH (1:20000; 60004‐1‐lg, Proteintech), P53 (1:20000;10442‐1‐AP, Proteintech), and p‐γH2Ax (S139) (1:1000; #9718, Cell Signaling), were diluted with 1% BSA/TBST and incubated with PVDF membranes overnight at 4 °C. The PVDF membranes were then incubated with HRP‐labeled goat anti‐rabbit (1:5000; Thermofisher) or anti‐mouse secondary antibodies (1: 5000; Thermofisher) diluted by 5% milk/TBST at room temperature for 1 h. The membranes were then subjected to ECL imaging using ECL kit (Vazyme, China), and images were captured with Tannon 5200SF at an auto mode.

Protein preparation, SDS-PAGE (using 4–12% gels; Invitrogen), transferred from the gel onto an Immobilon-PSQ transfer membrane (Millipore Corp, Bedford, MA) were performed at room temperature as previously described [18 (link),70 (link)]. Antibody labeling was detected by enhanced chemiluminescence (ECL) using primary antibodies: mouse monoclonal antibodies (Table 2). Anti-mouse secondary antibodies from Sigma-Aldrich and ECL kit from Pierce (Invitrogen) were used.