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. 2016 Jun 28;44(14):6853–6867. doi: 10.1093/nar/gkw571

Figure 2.

Figure 2.

Brd4 and SEC recruit P-TEFbs to DISF and NELF, respectively. (A) Cdk9-f immunoprecipitates with anti-Flag affinity resin from HSF of F1C2 (Cdk9-f) cells without or with HMBA treatment were analyzed with LC-MS/MS for Cdk9-associated proteins. The IPs from HeLa cells were served as negative control. The relative abundance of P-TEFb-bound proteins was calculated with NSAF method and the proteins with significant increase after HMBA treatment were shown. (B) The IPs were prepared as in (A) and analyzed by WB to confirm the interaction between P-TEFb and its associated factors as indicated in (A). The IPs from HeLa cells were served as negative control. (C) P-TEFb and its associated factors were purified with anti-Flag affinity resin from HSF of F1C2 (Cdk9-f) cells with shGFP (as control), shBrd4 or shAFF1+4 infection and HMBA treatment as indicated and analyzed by WB for the indicated proteins. (D and E) Anti-Flag immunoprecipitates from HSF of HeLa cells with indicated cDNA transfection and HMBA treatment were analyzed by WB for the indicated proteins.