Af596

HEK293 cells were infected with a multiplicity of infection (MOI) of 2 or 5 for 24–48 h with Ad5.CMV.sIL21R and Ad5.CMV.Null or Ad5.CMV.eGFP as negative controls. For immunocytochemistry, anti-IL21R antibody (AF596, R&D Systems, Minneapolis, MN, USA) and Alexa Fluor 488 donkey anti-goat secondary antibody (A11055, Invitrogen, Carlsbad, CA, USA) were used at a dilution of 1:100 and 1:200, respectively. For immunodetection, cell pellet was homogenized in lysis buffer (RIPA) and total protein was determined from lysed cells and supernatants using the Pierce BCA Protein Assay (232,227, Thermo Fisher Scientific, Waltham, MA, USA). Protein extracts and supernatants were loaded onto denaturing acrylamide gels and then electrotransferred to PDVF membranes (10,600,023, GE Healthcare, Chicago, IL, USA). Anti-IL21R primary antibody (AF596, R&D Systems) was used at 1:1000 dilution and goat HRP-anti-IgG secondary antibody (P0160, Dako, Agilent Technologies, Santa Clara, CA, USA) at 1:10,000 were used in the presence of 5% (w/v) of BSA (A9418, Sigma-Aldrich, St. Louis, MO, USA).