Journal: Scientific Reports
Article Title: SUMOylation of FOXP1 regulates transcriptional repression via CtBP1 to drive dendritic morphogenesis
Figure Lengend Snippet: FOXP1 is SUMOylated at K670. ( A ) DIV7 cortical neuronal cultures were lysed in buffer with or without 2% SDS and immunoblotted with FOXP1 antibody. Under denaturing conditions a prominent additional higher molecular weight FOXP1 band was observed. ( B ) Following lysis in 2% SDS containing buffer, the SDS was diluted to 0.1% and FOXP1 immunoprecipitated and incubated with GST, GST-WT-Senp1 catalytic domain or GST-Senp1-C603S catalytic domain. Immunoblots with FOXP1 antibody (top panel) shows the higher Mr band is removed by Senp1 treatment consistent with it being SUMOylated FOXP1. A high exposure (top left panel) of the input shows the presence of the modified-FOXP1. Lower panel shows equal levels of purified GST-SENP proteins by coomassie staining. ( C ) Immunoprecipitation was performed as in B, using a SUMO-1 antibody, followed by immunoblotting with either FOXP1 (upper panel) or SUMO1 (lower panel) antibodies. SUMO1-modified FOXP1 corresponds to the high Mr band in the input. Note the enrichment of SUMO1-ylated proteins (seen as a smear) in the IP lane but not the control IgG. ( D ) Schematic of FOXP1 showing the putative SUMOylated lysine. TRD, transcriptional repression domain; LZ, leucine zipper; FOX, FOX homolgy domain; NLS, nuclear localisation signal. ( E ) FOXP1 is SUMOylated at lysine 670. HEK293T cells were transfected with either TAP-FOXP1-WT or the mutant TAP-FOXP1-K670R, lysed in buffer containing NEM, to inhibit SUMO proteases, and precipitated using strepavidin beads before subsequent immunoblotting with anti-SUMO1 (top panel) or anti-SBP (lower panel) antibodies.
Article Snippet: Mass spectrometry HEK293T cells were transfected with either empty TAP vector or TAP-FOXP1 and lysed 48 hr after transfection in lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCL, 0.5% NP-40 and Roche protease inhibitor cocktail) before pull-down of the TAP tag using strepavidin beads (Sigma) for 1 hr.
Techniques: Molecular Weight, Lysis, Immunoprecipitation, Incubation, Western Blot, Modification, Purification, Staining, Transfection, Mutagenesis