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. 2015 Jun 19;10(6):e0129965. doi: 10.1371/journal.pone.0129965

Fig 4. SUMOylation of ΔLf represses its transcriptional activity.

Fig 4

A) Cells were co-transfected with pGL3-S1Skp1-Luc reporter vector and WT, SUMO mutant constructs or null vector in order to assay the relative transcriptional activity of WT and its SUMO mutants. Relative luciferase activities are expressed as described in Materials and Methods (n≥5; p < 0.05 (*)). B-E) Alteration of SUMOylation at K308 modulates ΔLf transcriptional activity. B) Knockdown of Ubc9 was performed using siUbc9/siCtrl as described in Materials and Methods and followed after 48 h of incubation by immunoblotting of the cell extracts with either anti-Ubc9 or anti-GAPDH antibodies. C) Knockdown of Ubc9 leads to a decrease in SUMOylation. Lysates were immunoprecipitated with M2 and immunoblotted with anti-SUMO-1 or M2. Input was immunoblotted with M2, anti-SUMO-1 or anti-GAPDH antibodies and used as controls. D) Deconjugation of SUMO-1 from WT, K13 and K308 by SENP2. HEK-293 cells were co-transfected with WT or the K308 construct together with pSG5-His-SUMO-1 and pcDNA-SENP2-SV5 and then lysed 24 h later. Lysates were immunoprecipitated with M2 and immunoblotted with anti-His. Input was immunoblotted with either M2 or anti-GAPDH antibodies. C-D) Cells were incubated with MG132 for 2 h prior to lysis and NEM added to lysis, IP and WB buffers. The data presented correspond to one representative experiment of at least six conducted (n ≥ 6) (B) and to one representative experiment of at least two conducted (n ≥ 2) (C, D). E) Cells were co-transfected with pGL3-S1Skp1-Luc reporter vector, either WT or the K308 construct together with pSG5-His-SUMO-1 or pcDNA-SENP2-SV5. Relative luciferase activities were also assayed in Ubc9 invalidated cells. HEK-293 cells were reverse transfected for 24 h using siRNAs targeting Ubc9 (siUbc9) or a scrambled control sequence (siCtrl) before being transfected as described above to evaluate the relative transcriptional activities of ΔLf and the K308 mutant. Relative luciferase activities are expressed as described in Materials and Methods (n≥5; p < 0.05 (*), p < 0.01 (**)).