Skip to main content
. 2020 Sep 8;117(38):23588–23596. doi: 10.1073/pnas.2004122117

Fig. 5.

Fig. 5.

TRIM28-PCNA interaction on human chromatin is dependent on RNAPIIo-associated RECQ5. (A) Western blot analysis of the indicated proteins in WCE prepared from HEK293T cells overexpressing the indicated FLAG constructs. Tubulin was used as a loading control. (B) Western blot analysis of the indicated proteins in CB:RNA+ (Left) and CB:RNA fractions (Right) of HEK293T cells overexpressing the indicated FLAG proteins. The ratio of the active form of RNAPII (RNAPIIo) relative to the inactive form of RNAPII (RNAPIIa) was used as the fractionation control for CB:RNA+ and CB:RNA- fractions. (C) Western blot analysis of the indicated proteins in WCE prepared from HEK293T cells with or without overexpression of FLAG-RECQ5 and treated with control or TRIM28 siRNA. (D) Western blot analysis of the indicated proteins in the CB:RNA+ fraction prepared from HEK293T cells with or without overexpression of FLAG-TRIM28 and treated with control or RECQ5 siRNA. (E) Diagram of human RECQ5. The SF2, KIX, PIP-L, and SRI domains are shown. Double arrows indicate protein–protein interactions among TRIM28, PCNA, RNAPIIo, and different domains of RECQ5. (F) Western blot analysis of the indicated proteins in WCE prepared from HEK293T cells with or without overexpression of FLAG-RECQ5 WT and mutants. (G) Western blot analysis of the indicated proteins in the FLAG-RECQ5 WT or mutant complexes immunopurified from CB fractions prepared from HEK293T cells with or without exogenously expressed FLAG-RECQ5 WT or mutant proteins as shown in F. (H) Western blot analysis of the indicated proteins in the CB fractions (Top) and the FLAG-TRIM28 complexes immunopurified from CB fractions (Bottom) prepared from HEK293T cells with or without exogenously expressed FLAG-TRIM28 and treated with control or RECQ5 siRNA. (I) Western blot analysis of the indicated proteins in the CB fractions (Top) and the FLAG-PCNA complexes immunopurified from CB fractions (Bottom) prepared from HEK293T cells with or without exogenously expressed FLAG-PCNA and treated with control or RECQ5 siRNA.