Figure 5. Acute bright-light stress does not reduce the steady-state level of Ub-charged UbcM2 or the catalytic activity of the enzyme. A: Lysates derived from the retinas of wt mice (lanes 1 and 2) and UbcM2^+/− mice (lanes 3–7) maintained in dim light (lanes 1, 3, 4, 5) or exposed to 3,000 lux for 6 h (lanes 2, 6, 7) were resolved by nonreducing (top blot) or reducing (bottom blot) sodium dodecyl sulfate PAGE (SDS–PAGE) followed by anti-UbcM2 western blotting. Ub-charged enzyme is evident as a slower migrating band in nonreducing SDS–PAGE (top blot, indicated by “UbcM2~Ub”). This band collapses to uncharged UbcM2 in samples exposed to reducing agent (bottom blot). Each lane represents lysate from an individual mouse. B: Left blot— Shown in this blot is the enzyme used in the auto-ubiquitylation assay. The enzyme was immunoprecipitated from UbcM2+/− retinal lysates. Each lane corresponds to lysate from an individual mouse. Lane 1 contains antibody (Ab) only to distinguish bands derived from the Ab versus those IPed by the Ab. Right blot—IPed UbcM2 was combined with recombinant E1, Ub, and energy and incubated at 37 °C for 90 min. A control lacking the auto-ubiquitylation reaction mixture is shown in lane 14. Reaction products were analyzed by SDS–PAGE and anti-UbcM2 western blotting. UbcM2-Ub₁ denotes a band that corresponds to mono-ubiquitylated UbcM2, and UbcM2-Ub₂ denotes a band that corresponds to two Ub molecules conjugated to the enzyme. Vertical lines between lanes 1 and 2 (left blot) and lanes 14 and 15 (right blot) indicate lanes were not adjacent on original blots. Molecular weight markers were run between samples 1 and 2 and samples 14 and 15. The migration of heavy chain is indicated, and the migration of molecular weight markers is shown on the left for (A) and (B).