FIG 4.
Free SUMO1 accumulation is allowed if conjugation to endogenous substrates is prevented or limited. (A) Western blot of F9 cells transduced with vectors expressing Flag-SUMO1 or Flag-SUMO1AA. (B) Western blot analysis of F9 cells engineered by successive transductions to coexpress Flag-SUMO1 (WT) or Flag-SUMO1ΔGG (ΔGG) along with HA-Trim28 (WT) or HA-Trim286KR (6KR), as indicated. Blots were probed with anti-Flag antibodies to detect SUMO1 and anti-HA antibodies to detect Trim28. The right four lanes show cells transduced with SUMO1 first and Trim28 second, and the left four lanes show cells transduced with Trim28 first and SUMO1 second. Prior expression of Trim28 allowed good expression of WT SUMO1; subsequent expression was not effective. (C) Western blot analysis of F9 cells engineered by successive transductions to coexpress Flag-SUMO1 (WT) or Flag-SUMO1ΔGG (GG) along with Myc-RanGAP1. Blots were probed with anti-Flag antibodies to detect SUMO1 and with anti-Myc antibodies to detect RanGAP1. The right two lanes show cells transduced with SUMO1 first, and the left two lanes show cells transduced with RanGAP1 first. Prior expression of RanGAP1 allowed good expression of WT SUMO1; subsequent expression was not effective. (D) Western blot analysis of F9 cells coexpressing Flag-SUMO1 and HA-SENP1. The blot was probed with anti-Flag antibodies to detect SUMO1, and with anti-HA antibodies to detect SENP1. The left two lanes show cells transduced with SENP1 first, and the right two lanes show cells transduced with SUMO1 first.