Fig. 5

DDX39B is modified by SUMO2 following TMZ treatment. a Nickel column (Ni-NTA) pull-down in HeLa cells expressing empty vector, 6His-Sumo1 or 6His-Sumo2. Immunoblot (IB) with anti-DDX39B. Input lysate also probed with anti-DDX39B or anti-GAPDH control. b Co-immunoprecipitation (Co-IP) in 293T cells transfected with S-tag DDX39B and HA-Sumo1, HA-Sumo2, or HA-Sumo3. IP performed with S-agarose and IB with anti-HA. Input lysate was probed as shown. c Co-IP in U87 cells treated with TMZ (100 μM) for the indicated time. IP with anti-DDX39B or IgG control and IB with anti-SUMO and anti-DDX39B. Input (lower blot) was probed as shown. d Schematic of predicted Sumo sites in DDX39B. DEAD-box domains indicated. e Co-IP in 293T cells transfected with empty vector (EV) or S-tag DDX39B (wild-type or the indicated mutant) and HA-Sumo2. IP performed with S-agarose followed by IB with anti-HA. f Ni-NTA pull-down in HeLa-SUMO2 cells transfected with EV or FLAG-DDX39B (wild-type or mutant) as indicated and treated with vehicle or TMZ (100 μM, 12 h). IB with anti-FLAG. g Ni-NTA pull-down in HeLa-SUMO2 cells transfected with empty vector or the indicated FLAG-PIAS protein. IB performed with anti-DDX39B. Input lysate was probed with the indicated antibody. Asterisk, sumoylated band. h Co-IP in U87 cells transfected with empty vector or Flag-PIAS proteins as indicated. IP with anti-FLAG and IB with anti-DDX39B. Input was probed with the indicated antibody. i Co-IP in U87 cells treated with vehicle or TMZ (100 μM, 12 h). IP performed with anti-PIASx or IgG control and IB with anti-DDX39B antibody. j IB in U87 cells transfected with si-PIASx or si-control and treated with vehicle or TMZ (100 μM, 12 h). IP performed with anti-DDX39B and IB with anti-SUMO