G box chemi detection system

Cell lysates were obtained using lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 0.5% NP-40, 10% glycerol, and protease and phosphatase inhibitor cocktail sets (Calbiochem)). Immunoprecipitations were performed with protein A/G magnetic beads (Millipore) mixed with immunoprecipitation antibodies. After overnight incubation at 4°C, beads were washed with high salt lysis buffer (containing 300 mM NaCl), boiled in SDS sample buffer (Boston BioProducts), resolved by SDS-PAGE (Criterion TGX precast gels, Bio-Rad), transferred to nitrocellulose membranes (Bio-Rad), blocked and incubated with the appropriate primary antibody in TBS-T overnight at 4°C. Detection of proteins was performed with horseradish-peroxidase conjugated secondary antibodies (Rockland), developed using Clarity Western ECL substrate (Bio-Rad), and imaged with a G:BOX Chemi detection system (Syngene).

The following antibodies were used: MCPyV Ab5 [36 (link), 57 (link)]; GFP (D5.1, Cell Signaling); vinculin (H-10, Santa Cruz); actin (D6A8, Cell Signaling); HK2 (C64G5, Cell Signaling); LDHA (EP1565Y, Abcam); MCT1 (A1512, NeoBiolab); LAT1 (5347, Cell Signaling); RelA (D14E12, Cell Signaling); IκBα (L35A5, Cell Signaling); MYC (9E10, Santa Cruz); MYCN (9405, Cell Signaling); MYCL (AF4050, R&D Systems).
Whole cell lysates were prepared using RIPA buffer (Boston BioProducts) supplemented with protease inhibitor cocktail set I (Calbiochem) and phosphatase inhibitor cocktail set I (Calbiochem). Clarified protein extracts were boiled in SDS sample buffer (Boston BioProducts), resolved by SDS-PAGE (Criterion TGX precast gels; Bio-Rad), transferred to nitrocellulose membranes (Bio-Rad), blocked and incubated with the appropriate primary antibody in TBS-T overnight at 4°C. Detection of proteins was performed with horseradish peroxidase-conjugated secondary antibodies (Rockland), developed using Clarity Western ECL substrate (Bio-Rad), and imaged with a G:BOX Chemi detection system (Syngene).