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. 2012 Sep;32(17):3428–3437. doi: 10.1128/MCB.00247-12

Fig 5.

Fig 5

Cyclin T1 and Fcp1 double knockdown increases Pol II levels at the 5′ end of Hsp70 during heat shock. (A) Western blots of whole-cell extracts from untreated, LacZ-RNAi, cyclin T1-RNAi, Fcp1-RNAi, and double knockdown cyclin T1-Fcp1-RNAi cells probed with antibodies for cyclin T1 (1:1,000; lab stock), Fcp1 (1:1,000; lab stock), and TFIIS (1:3,000; lab stock loading control). The relative amount loaded is indicated (where 1 = 1.5 × 106 cells). (B) ChIP results for the Pol II subunit Rpb3 enrichment on the Hsp70 gene in untreated, LacZ-RNAi, cyclin T1-RNAi, Fcp1-RNAi, and double knockdown cyclin T1-Fcp1-RNAi cells at 10 min of heat shock. The x axis shows the midpoint of each PCR fragment along the Hsp70 gene, and the y axis shows the percentage of input DNA immunoprecipitated (error bars indicate the standard error of the mean of at least four biological replicates). (C) Phosphorylated CTD serine 5 (3E8 at 1:250; EMD Millipore), serine 2 (3E10 at 1:250; EMD Millipore), and TFIIS (1:3,000; lab stock loading control) Western blots of nonchromatin (free) fractions from untreated, LacZ-RNAi, cyclin T1-RNAi, Fcp1-RNAi, and double knockdown cyclin T1-Fcp1-RNAi cells. The relative amount loaded is indicated (where 1 = 1 × 106 cells).