EGR1 induced by EGF is sumoylated and ubiquitinated in vivo.

(A) ECV304 cells were treated for 1 hour with 10 ng/ml EGF to induce Egr1 expression. Lysates were immunoprecipitated with polyclonal antibody to EGR1 and immunoblotted with anti EGR1 and, after stripping, with anti SUMO-1 antibodies. (B) ECV304 cells were treated 1 hour with 10 ng/ml EGF in the absence or in the presence of 100 µM NEM and lysates obtained were immunoprecipitated with a polyclonal antibodies to EGR1 and immunoblotted with antibodies anti EGR1. The arrows indicate bands corresponding to EGR1 and EGR1 plus SUMO-1. (C) ECV304 cells were treated for 15, 30 or 60 minutes with 10 ng/ml EGF. Lysates were immunoprecipitated with polyclonal antibody to EGR1 and immunoblotted with anti EGR1 and, after stripping, with anti K48 ubiquitin antibodies. (D) ECV304 cells were treated for different times with 10 ng/ml EGF in the absence or in the presence of 100 µM NEM and lysates obtained were resolved by SDS-PAGE and analyzed by Western blot with antibodies against EGR1 and tubulin. All experiments are representative of three independent.