Figure 2. SENP6 regulates SUMOylation of RPA70.

(A) SENP6-knockdown induces RPA70 SUMOylation. Cell lysates were prepared at 72hr after transfection with indicated siRNAs, separated by SDS-PAGE, and immunoblotted with anti-RPA70 monoclonal antibodies. Two different exposures were shown.

(B) Endogenous RPA70 is modified by endogenous SUMO-2/3 in vivo. SENP6-knockdown was described in Methods. Cell lysate was aliquoted equally for performing immunoprecipitation by control IgG or RPA70 monoclonal antibody. Endogenous SUMOylated RPA70 was immunoblotted with anti-SUMO-1 or anti-SUMO-2/3 antibodies. Loading samples were standardized by blotting with anti-RPA70 monoclonal antibodies.

(C) RPA70 is modified by SUMO-2 in an over-expression system. COS-1 cells were transfected with indicated c-Myc-tagged RPA70 (2.5 ug) and HA-tagged SUMO-2 (0.5 ug) plasmids. Immunoprecipitation was performed with Myc-EZview™ Red Affinity Gel matrix 24hr after transfection and analyzed with c-Myc (upper panel) or HA antibodies (lower panel).

(D) Over-expression of SENP6 de-conjugates SUMOylated RPA70. COS-1 cells were transfected with c-Myc-tagged RPA70 (2.0 µg), HA-tagged SUMO-2 (0.5 µg), and SENP6 (0.5 µg) or SENP6 catalytic site mutant (0.5 µg) plasmids. Tagged RPA70 was precipitated and immunoblotted with anti-HA (upper panel) or anti-c-Myc antibodies (middle panel). SENP6 expression was confirmed by immunoblotting with anti-FLAG antibodies (lower panel). Over-expression of SENP6 may result in non-specific cleavage. However, the true specificity of SENP6 was shown by the siRNA knock down studies shown in Figure 2A and B.

(E) Determination of SUMOylation sites in RPA70. COS-1 cells were transfected with 2.0 µg of c-Myc-tagged RPA70 or its mutants along with HA-SUMO-2 plasmids. Immunoprecipitates of c-Myc-tagged RPA70 were immunoblotted with anti-HA (upper panel) or anti-c-Myc antibodies (lower panel).