Figure 5. The CFIm68/59 RS-like domain binds to Fip1.
(A) HeLa nuclear extract (NE) or recombinant CFIm25-68 or CFIm25-59 complexes purified from baculovirus-infected Sf9 insect cells with or without alkaline phosphatase (CIP) treatment, were resolved by Phos-tag gel and analyzed by western blotting. The red arrows point to the phosphorylated proteins and the green arrows dephosphorylated proteins. (B) CFIm59 and CFIm68 RS domain sequences. (C) GST pulldown assay with GST, GST-RS(CFIm59) or (CFIm68) (purified from Sf9 cells) and in vitro translated 35S-labeled individual CPSF and CstF subunits. GST pulldown samples were resolved on SDS-PAGE and visualized by phosphorimaging (top panel). The same pulldown assay was performed with 6xHis-Fip1 expressed in Sf9 cells and pulldown samples were resolved on SDS-PAGE and analyzed by western blotting (lower panel). (D) A diagram of the Fip1 domain/regions. The Fip1-N and -C fragments were marked. Pulldown assays were similar to (C) with in vitro translated and 35S-labeled Fip1-N and Fip1-C. (E) Top panel: the sequences of the Fip1-RD and -RA peptides. Lower panel: Fip1-RD and –RA pulldown with purified 6xHis-CFIm25 (E. coli), 6xHis-CFIm25-59 (Sf9), and 6xHis-CFIm25-68 complexes (Sf9) and the bound proteins were resolved on SDS-PAGE and analyzed by western blotting. Negative control: streptavidin beads (beads). (F) Top panel: GST-RS(CFIm59/68) purified from E. coli were mock treated (−) or treated (+) with SRPK1 and then used in pulldown assays with 6xHis-Fip1. The pulldown samples were analyzed by western blotting. Lower panel: GST-RS(CFIm59/68) purified from Sf9 cells were mock untreated (−) or treated (+) with CIP, and then used in pulldown assays with purified 6xHis-Fip1. (G) Nuclear extracts from control, CFIm59-KO, or CFIm68-KO HEK293T cell lines were used for IP with anti-CFIm25 antibody and the IP samples were analyzed by western blotting. The red arrows mark the CFIm59 or CFIm68 that are absent in KO cell lines.