Sds page sample loading buffer

All chemicals were of an analytical grade. Water, methanol (MeOH), formic acid, and acetonitrile (LC/MS grade Optima), chloroform, Pierce™ Trypsin Protease MS Grade, Pierce™ DTT (Dithiothreitol), Bolt^® MOPS SDS Running Buffer (20×), mini protein gel NuPAGE™ 4 to 12% Bis-Tris, and 4X Bolt™ LDS Sample Buffer were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM), trypsin, glutamine, phosphate-buffered saline (PBS), and Foetal Bovine Serum (FBS) were obtained from Lonza^® (Verviers, Belgium). Ethanol (96%) was purchased from Carlo Erba (Peypin, France). Iodoacetamide and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (Barcelona, Spain). Tris(hydroxymethyl)aminomethane and glacial acetic acid were obtained from Merck Milipore^® (Burlington, MA, USA, EUA). Coomassie Brilliant Blue R-250 was purchased from BIORAD^® (Hercules, CA, USA). A 5× SDS-PAGE Sample Loading Buffer and NZYBlue Protein Marker were purchased from Nzytech^® (Lumiar, Portugal).

Transduced cells were harvested and lysed in ice-cold Pierce IP Lysis Buffer (Thermo Scientific, #87787) at 4° C. Cell lysates were mixed with 5× SDS-PAGE Sample Loading Buffer (Nzytech, MB11701), and heated at 95° C for 5 min. Protein samples were resolved by SDS polyacrylamide gel electrophoresis and transferred onto a PVDF membrane using Mini Trans-Blot System (Bio-Rad, #1703935), followed by blocking for 1 h with 5% BSA in Tris-buffered saline-Tween20 buffer and probing with corresponding primary and secondary antibodies (Table S5). The proteins were visualized by chemoluminiscence using ChemiDoc Imaging Systems (Bio-Rad). Relative protein expression was calculated by sequentially normalizing against the loading control (GAPDH).