Figure 5

TgCdk7 phosphorylates TgRPB1-CTD at serine 5 of the heptapeptide repeats.

(A) Schematic diagram of TgRPB1. The carboxyl-terminal domain (1631–1847 aa) containing putative nine heptad repeats of YSPxSPx is depicted. (B,C) Polyclonal antibody raised against recombinant TgRPB1-CTD recognized the recombinant (~27 kDa) and native proteins (~209 kDa) at the expected sizes in a concentration dependent manner. (D) IF analysis using anti-TgRPB1 antibody showed punctate nuclear foci in the parasite. Scale bar 5 μm. (E) Phosphorylation of Ser5 residue of heptapeptide repeats in TgRPB1 was demonstrated using IP with anti-TgRPB1 antibody followed by λ-phosphatase treatment and Western blot analysis using P-Ser5 antibody. No band was detected in the pre-immune IP. Similar levels of TgRPB1 in lanes 1 and 2 suggested equal pull down of TgRPB1. (F) Inhibition of TgCdk7 by BS-181 followed by TgRPB1 IP and western blot using P-Ser5 antibody and P-Ser2 antibody. Western blot shows abrogation of P-Ser5 while P-Ser2 is also affected. No inhibitory effect was observed for DMSO treated parasite. No band was detected in the pre-immune IP. Similar levels of TgRPB1 in lanes 1, 2, 3 and 4 suggested equal pull down of TgRPB1. Similar level of TgRPB1 (bottom panel E,F) indicates equal amount of parasite proteins used for IP.