Pull-down assays showing the interaction of His-tagged PRPF31 (WT and A216P mutant) with GST-tagged PRPF6 domains

Complexes between the two proteins were immobilized with glutathione sepharose beads and eluted with reduced glutathione. Panels show the Western analysis of elution products using immobilized GST-PRPF6N (amino acids 1–306), GST-PRPF6M (amino acids 307–607), and GST-PRPF6C (amino acids 607–941). Upper panels were probed with α-His.tag antibody and lower panels with α-GST antibody. Asterisks (*) indicate the size of full-length GST-PRPF6N, GST-PRPF6M, and GST-PRPF6C respectively. Note that the faint band in the upper panel for the pull-down assay with GST-PRPF6N is too small for PRPF31-His. Negative controls show absence of nonspecific binding of His-tagged PRPF31 to the beads and to GST tag. Quantification of the WT and mutant PRPF31 bands gave ratios of mutant:WT of 4.2±1.4 (for PRPF6M) and 2.1±0.5 (for PRPF6C). In each case, the mean and standard error was determined from four separate determinations.<p><b>Copyright information:</b></p><p>Taken from "Disease mechanism for retinitis pigmentosa (RP11) caused by missense mutations in the splicing factor gene "</p><p></p><p>Molecular Vision 2008;14():683-690.</p><p>Published online 18 Apr 2008</p><p>PMCID:PMC2324120.</p><p>