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. 2008 May 9;7(7):1158–1167. doi: 10.1128/EC.00434-07

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FIG. 4. — (A) The loss of some Paf1C components reduces Pol II CTD Ser2-P. Protein extracts prepared from isogenic strains bearing the indicated mutations were separated by electrophoresis, blotted, and probed for the indicated antigens as described in Materials and Methods. The strains used were YJJ662 (WT), YJJ1821 (ctk1Δ), YJJ577 (paf1Δ), YJJ1197 (ctr9Δ), YJJ665 (cdc73Δ), YJJ1303 (rtf1Δ), and YJJ1336 (leo1Δ). Antibodies used for Pol II were H5 (α-Ser2-P), H14 (α-Ser5-P), and 8WG16 (α-unphosphorylated CTD [CTD unphos.]). (B) The reduction of Pol II CTD Ser2-P by the loss of Paf1 is not yeast strain dependent. Protein extracts from pairs of isogenic strains with the indicated genetic backgrounds were analyzed for the Pol II Ser2-P CTD as described in the legend to panel A by using the BL2894 antibody. The strains used were YJJ662 (WT; lane 1), YJJ664 (paf1Δ; lane 2), YSB2116 (WT; lane 3), YSB2118 (paf1Δ; lane 4), and YJJ1871 (ctk1Δ; lane 5). (C) Schematic of the MAK21 gene showing the locations of primers spanning the first poly(A) site [P(A)1] used for RT-PCRs. (D) The loss of some Paf1C factors, but not Leo1 or Ctk1, increases the read through of the MAK21 poly(A) site. RT-PCR was used to measure the amount of the MAK21 read-through transcript, which was normalized to the signal from the PMA1 gene. Strains used for RNA isolation were the same as those listed in the legend to panel A. (E) The loss of Rtf1 or Cdc73 does not affect the abundance of HA-tagged Ctk1. Protein extracts from isogenic HA-tagged Ctk1 strains were analyzed as described in the legend to panel A. The strains used were YJJ1873 (WT), YJJ1875 (rtf1Δ), YJJ1897 (cdc73Δ), and YJJ1882 (paf1Δ). Extracts in the upper panel were probed with α-HA. Those in the lower panel were probed with α-G6PD. (F) The loss of Rtf1 or Cdc73 does not affect the abundance of HA-tagged Cft1. Protein extracts from isogenic HA-tagged Cft1 strains were analyzed as described in the legend to panel A and probed with α-HA. The strains used were YJJ1864 (WT), YJJ1850 (cdc73Δ), and YJJ1849 (rtf1Δ).