PRPF40B associates with the splicing factors SF1 and U2AF65 in vitro. (A) Schematic representations of the GST-PRPF40B fusion proteins are shown. The numbers in parentheses represent the PRPF40B amino acids contained in the constructs. The two WW domains and five FF domains are indicated. (B) Both the amino- and carboxyl-terminal portions of PRPF40B are important for interacting with SF1. Recombinant full-length GST-PRPF40B and its amino- and carboxyl-terminal regions were bound to glutathione–agarose beads and incubated with T7-tagged SF1 partially purified from HEK293T. GST and GST-TCERG1 carboxyl terminus (Ct) were used as negative controls, and the GST-TCERG1 amino-terminal region (Nt) was the positive control. Eluted proteins were separated by SDS-PAGE and analyzed by Western blotting (top) using anti-SF1 to assess the interactions with SF1 or silver staining (bottom) to detect the GST fusion proteins. (C) GST, GST-U2AF65, and GST-PRPF40B were used to bind recombinant SF1. The bound protein was eluted with a linear gradient of 150–500 mM NaCl. After the elution step, the samples were separated by SDS-PAGE and analyzed by Western blotting or silver staining as described above.
