Fig 3. Competition between SUMOylation and ubiquitination at K379 controls ΔLf turnover.

A) HEK-293 cells were co-transfected with K379 or NV constructs, His-SUMO-1 or/and HA-Ub-expression vectors for 24 h and then incubated with 10 μM of the proteasomal inhibitor MG132 for 2 h prior to lysis. NEM was added to lysis, IP and WB buffers. Total cell extracts were immunoprecipitated with M2 or used as input. Samples were immunoblotted with anti-HA (upper panel) or with anti-SUMO-1 (lower panel) antibodies. Input was immunoblotted with either M2 or anti-GAPDH antibodies and used as loading control. NS: non-specific. The data presented correspond to one representative experiment of at least three conducted (n ≥ 3). Lane 6 corresponds to non-transfected cells. B) Cells were transfected with K379, either with the His-SUMO-1 or the HA-Ub expression vector and then incubated with fresh medium supplemented by 10 μg.mL^-1 CHX for the indicated time 24 h after transfection. K379 transfected cells were incubated without (left panel) or with (right panel) 10 μM MG132 for 2 h prior to lysis. Total protein extracts were immunoblotted with either M2 or anti-GAPDH antibodies. Detection was carried out using a Fusion SOLO camera (Vilbert Lourmat). The data presented (B) correspond to one representative experiment of at least five conducted. C-D) The M2 densitometric analyses are normalized for the matching GAPDH immunoblots and expressed as ratio D^K379/D^GAPDH as described in Materials and Methods. Data are shown as the means ± SD (n = 5).