Fig. 7

TRIP12 ubiquitylates BRG1 but does not affect its stability.A HEK293FT cells were transfected with Myc-TRIP12 with or without Wnt3a and MG132 treatment. In A and B, ubiquitylated proteins were pulled down with MBP-TUBE. BRG1 and TRIP12 were detected by immunoblotting. MBP-TUBE and α-tubulin are loading controls. B HEK293FT cells transfected with control vector, Myc-TRIP12 wild-type (TRIP12) or Myc-TRIP12 catalytic dead (TRIP12-CD) and FLAG-BRG1 and treated with Wnt3a and MG132. C HEK293FT cells were transfected with nontargeting (siNT) or two pooled TRIP12 siRNAs and control vector or HA-Ubiquitin (HA-Ub). Cells treated with Wnt3a and MG132 and extracts immunoprecipitated with BRG1 antibody or immunoglobulin G (IgG). Ubiquitylated BRG1 detected with HA antibody. D Recombinant BRG1 incubated with GST-TRIP12-HECT domain. Ubiquitylated BRG1 was detected by immunoblotting. E–L yellow (y) control or ctrip RNAi expressed in the dorsal compartment of larval wing discs using apterous (ap)-Gal4. E, H, K, L ap-Gal4-driven expression of y RNAi control. No change observed in Bap111-GFP (green) or Brm-GFP (green) levels. F, G, K ap-Gal4-driven expression of ctrip RNAi increased Bap111-GFP (green) levels in the dorsal compartment. I, J, L ap-Gal4-driven expression of ctrip RNAi in the dorsal compartment results in no change in Brm-GFP levels (green). Arrows denote the dorsal-ventral boundary. K, L Quantification of GFP mean intensity as dorsal-ventral ratio. Bap111 level increased in the dorsal compartment upon Ctrip knockdown and quantified K. Graphs show mean ± SEM, n = 9, 9, 14 in K, n = 12, 11, 9 in L. N is the number of wing discs analyzed. Significance assessed using one-way ANOVA with Dunnett’s test, comparing genotypes to control (yi1). p-values for K are both <0.0001. p-values for L are 0.1749 and 0.1986. Scale bar (E–J): 50 μm. Dorsal, top; posterior, right. M HEK293FT cells transfected with empty vector or Myc-TRIP12, treated with recombinant Wnt3a, and immunoblotted for TRIP12 and BRG1. *indicates nonspecific band in M and N. N HEK293FT cells transfected with nontargeting (siNT) control or TRIP12 siRNAs, treated with recombinant Wnt3a. Lysates were immunoblotted for endogenous TRIP12 and BRG1. α-tubulin is a loading control. All immunoblots represent at least three independent experiments. ****p < 0.0001, p ≥ 0.05 is not significant (ns). Source data provided.