FIG. 4.

Absence of SUMO-1-conjugated proteins and free SUMO-1 in MEF and testis lysates of wild-type and Sumo-1-null mice. Proteins in cell lysates were resolved by electrophoresis on 4 to 20% polyacrylamide gradient gels under denaturing conditions and transferred to nitrocellulose membranes. Panels A to C show immunoblots (IB) with anti-SUMO-1 antibody (A), anti-SUMO-2 antibody (B), and anti-VP16 antibody (C). In panel A, the narrow strip shows the free SUMO-1 region after a 10-fold longer exposure than for the main immunoblot. Anti-β-tubulin IB is shown for comparison in each instance. Asterisks depict the ∼90-kDa band corresponding to sumoylated RanGAP1; arrowheads mark free SUMO-1, and the arrow identifies free SUMO-2.