Figure 2: Gcm^R59L proteins are hyperubiquitinated.

(a) Western blots showing expressions of Gcm and Gcm^R59L in S2 cells in the absence or presence of MG132 (50 μM). Lysates were collected at 4 h after MG132 treatment. Quantifications for relative band intensity of Flag to α-Tubulin were shown at the bottom. (b,c) Co-IP analysis of cells co-transfected with p3xHA-Ub and p3xFlag-gcm or p3xFlag-gcm^R59L. Ubiquitinated Gcm and Gcm^R59L were detected as smears using anti-HA antibody after immunoprecipitation with anti-Flag antibody. Quantifications for relative band intensity of HA to Flag were shown at the bottom. Inputs were shown in (c). Note that a much higher ubiquitination level was detected for Gcm^R59L. (d,e) Co-IP analysis of S2 cell lysates co-transfected with p6xMyc-Slimb and p3xFlag-gcm or p3xFlag-gcm^R59L indicated that both Gcm and Gcm^R59L bind Slimb. Quantifications for relative band intensity of Myc to Flag were shown in (d). Inputs were shown in (e). (f) Gcm^R59L binds DNA. ChIP assays were performed using S2 cell lysates transfected with p3xFlag-gcm or p3xFlag-gcm^R59L in absence or presence of MG132 (50 μM). For quantification, results were normalized against the level for Gcm without MG132 treatment (designated as 1.0). Each ChIP experiment was repeated three times (n = 3). Note a drop in the fold of binding enrichment when cells are transfected with p3xFlag-gcm^R59L. No significant difference was detected for cells transfected with p3xFlag-gcm or p3xFlag-gcm^R59L in the presence of MG132. Averages are mean ± standard error of mean (SEM), *represents p < 0.05, **represents p < 0.01, and ***represents p < 0.001 by Student’s t test. ns means no significance. Western blot gels have been run under the same experimental conditions.