Xcell iitm blot module

BN‐PAGE gels were transferred to PVDF membranes in Dunn carbonate buffer (10 mM NaHCO₃, 3 mM Na₂CO₃) applying a constant voltage of 25 V at 4°C for 1 h using a XCell II^TM Blot Module (Thermo Fisher Scientific). SDS‐PAGE gels were transferred to PVDF membranes in Tris‐Glycine transfer buffer (25 mM Tris‐HCl, 192 mM Glycine, 20% methanol, 0.025% SDS) applying a constant voltage of 100 V at 4°C for 1 h using a Mini Trans‐Blot® Cell (Bio‐Rad). For the immunodetection of specific protein targets, blotted PVDF membranes were blocked in 5% skimmed milk in PBS‐T (0.1% Tween‐20) at room temperature for 1 h and then incubated overnight with primary antibodies diluted in 3% BSA in PBS‐T overnight at 4°C. After incubating the primary antibody, the PVDF membranes were washed three times with PBS‐T for 10 min, incubated with the secondary HRP‐ conjugated antibody for 1 h at room temperature and washed three times with PBS‐T for 10 min. Chemiluminescent signals were recorded using an Alliance Mini HD9 (UVITEC). Antibodies used are listed in the table of antibodies (Table 2).

Osteosarcoma cells were lysed using high-salt buffer (20 mM Tris*HCl, 400 mM NaCl, 0.5% NP40, 0.3% Triton X100) with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). The protein content was assessed using the Bio-Rad Protein Assay Kit II (Bio-Rad, Hercules, CA, USA). After denaturing the samples in NuPAGE^® LDS Sample Buffer (Thermo Fisher Scientific), 25 µg proteins were separated by electrophoresis using NuPAGE^® 4–12% Bis-Tris Protein Gels (Thermo Fisher Scientific), transferred onto Immobilon-FL PVDF membranes (Merck) with the XCell IITM Blot Module (Thermo Fisher Scientific), and treated with Odyssey Blocking Buffer TBS (LI-COR). Antibodies directed against the following targets were used to probe the membranes: phospho-histone H2A.X (Ser139) (D7T2V) mouse monoclonal antibodies (Cell Signaling Technology) and GAPDH (Abcam), dilutions 1:1000 and 1:2500 respectively. The DyLight^™ conjugated antibodies (Cell Signaling Technology) were used diluted 1:20,000 for visualization of specific bands, which was made on the Odyssey Imaging System (Li-COR). Quantification was performed in Image Studio Lite 5.2 (Li-COR).