Fig. 5: Auto-sumoylation of Ubc9 is enhanced in ulp2Δ 500 G (Uba2C162S) cells.
From: Auto-sumoylation of the yeast Ubc9 E2 SUMO-conjugating enzyme extends cellular lifespan
a Immunoprecipitation (IP) of Ubc9-Flag with anti-Flag agarose from denatured yeast extracts in the indicated strains expressing Flag-tagged Ubc9 followed by immunoblot analysis with anti-SUMO or anti-Flag antibodies. Anti-Pgk1 was used as a loading control for protein input. Arrowheads, open circles, and asterisks indicate sumoylated Ubc9, unsumoylated Ubc9, and nonspecific bands, respectively. b Growth of the ubc9-K153/157 R (ubc9-RR) mutants from two different strain backgrounds (W303 and MHY500). After spotting cells in fivefold serial dilutions, the YPD plates were incubated for 2 days at 30 °C. c, f, g Immunoblot assay of sumoylated proteins in extracts prepared from the indicated strains. The plus and minus symbols represent the presence and absence of YCplac33-ULP2, respectively, in the indicated uba2, ubc9, and ulp2 mutants. Anti-Pgk1 blotting was used to verify equal loading. The stacking gel (bracket) and molecular size standards (in kDa) are indicated. d, e, h RLS analysis of indicated strains, as shown in Fig. 3a.
