Fig. 3

Functional analysis of SAP130 sumoylation. A Repression of a Gal4-dependent luciferase reporter gene relative to Gal4 control by Gal4 fusions to SAP130 WT and 3KA mutant. B FAF1, SAP130 WT and 3KA mutant efficiently inhibited mineralocorticoid receptor (MR) transactivation. The MMTV-Luc reporter plasmid, the internal control plasmid pRL-TK (Renilla), pRS-hMR, and two different doses of the HA-tagged-FAF1, SAP130 WT or 3KA mutant plasmids were co-transfected into COS-1 cells. The total DNA amount was 2 μg (including addition of empty vector). At 24 h post-transfection, cells were treated with either vehicle or 1 μM aldosterone (Aldo.), and reporter gene activities were measured after another 24 h period. C Both SAP130 WT and 3KA mutant could inhibit Wnt signaling. HEK293 cells were co-transfected with the TopFlash-Luc reporter, pRL-TK, and HA-FAF1 or SAP130 WT or 3KA mutant plasmids. Cells were treated with Wnt3A conditioned medium (CM) or left untreated and lysed for a luciferase assay at 48 h post-transfection. D Both SAP130 WT and 3KA mutant could efficiently inhibit NF-κB activation. HEK293T cells were co-transfected with pNF-κB-Luc reporter plasmid and pRL-TK plasmid together with two different doses of HA-tagged-FAF1, SAP130 WT or 3KA mutant plasmid. At 36 h post-transfection, cells were treated with tumor necrosis factor (TNF)-α (20 ng/ml) for 6 h or left untreated, and then were subjected to a luciferase assay. E SAP130 and FAF1 cooperate in repressing gene transcription. HEK293T cells were transiently transfected with pNF-κB-Luc, SAP130, and FAF1 expression plasmids or infected with lentiviruses expressing control shRNA or FAF1 shRNA as indicated. At 36 h post-transfection/post-transduction, cells were treated with TNF-α (20 ng/ml) for 6 h or left untreated, and followed by a luciferase assay. F The SUMO mutation does not alter SAP130 subcellular localization. Immunofluorescence images of HeLa cells transiently expressing HA-tagged SAP130 WT or 3KA as indicated. Immunostaining was performed with an anti-HA antibody (green). DAPI staining shows the position of the nucleus. Scale bars represent 10 μm. G Effect of the proteasome inhibitor MG132 on the accumulation of SUMO1-modified and unmodified forms of SAP130. HA-SAP130 WT and 3KA SAP130 mutant were co-transfected with EGFP control or EGFP-SUMO1 plasmid into COS-1 cells. Twenty-four hours after transfection, the cells were cultured in the presence and absence of 5 μM MG132 for another 8 h. SAP130 accumulation was detected by WB with an anti-HA antibody. Cell lysates were also immunoblotted with anti-EGFP antibody to confirm the expression of EGFP and EGFP-SUMO1. The bracket and arrow indicate EGFP-SUMO-1-modified and unmodified SAP130 proteins, respectively. The blue and red arrowheads indicate the EGFP and EGFP-SUMO1 proteins, respectively. H Cell growth curves of HEK293 cells stably transfected with constructs expressing control vector, HA-SAP130 WT or 3KA. SAP130 expression was analyzed using immunoblotting. I Colony formation assays were performed in stable vector (control), SAP130 WT and SAP130 3KA-overexpressing cells. Colonies were stained with 2% methylene blue 14 days later. Quantitative results in A-E, H and I represent the mean ± SD of three independent experiments. Statistical analyses were performed using two-tailed Student’s t-test. *p < 0.05; **p < 0.01. The immunoblots were cropped for clarity. Full length blots are presented in Supplemental Figure S10