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. Author manuscript; available in PMC: 2012 May 20.
Published in final edited form as: Mol Cell. 2011 May 20;42(4):477–488. doi: 10.1016/j.molcel.2011.03.011

Figure 4. RNF20 knockdown increases the occupancy of TFIIS on chromatin.

Figure 4

(A) Western blot analysis of RNF20, TFIIS, H2Bub and H2B 48 hours after transient transfection of HeLa cells with the indicated siRNA oligonucleotides. Equal amounts of total protein were loaded in each lane.

(B) HeLa cells were transiently transfected with the indicated siRNA oligonucleotides. 48 hours later, the chromatin bound fraction was isolated as described under Experimental procedures, and subjected to Western blot analysis with the indicated antibodies. Whole cell extract aliquots (Total) were analyzed in parallel.

(C) Chromatin immunoprecipitation (ChIP) analysis of relative TFIIS abundance on RNF20-suppressed genes (NR4A2, RhoB, FOS, FOSL2, CASP9, HBA1), the RNF20-independent gene GAPDH and the RNF20-dependent gene H2BD, in siLacZ and siRNF20-transfected HeLa cells. Immunoprecipitated DNA and input DNA were quantified by qPCR with primers specific for the 5′ transcribed regions of the indicated genes. Results are presented as percentage of input. Bars indicate averages of data from duplicate qPCR reactions; error bars represent standard deviation. Similar data was obtained in 3 independent experiments.