Figure 3. SCF Slimb-mediated ubiquitination of PAR-1 is coupled to its phosphorylation at T408.

(a) Western blot analysis showing enhanced in vivo ubiquitination of PAR-1 by Slimb. (b) Western blot analysis showing effects of Slimb-WT or Slimb-ΔF on transgene-derived total PAR-1 and p-PAR-1 levels. Actin serves as a loading control. (c) Quantification of the ratio of p-PAR-1/total PAR-1 levels shown in b. (d) IP experiment showing selective binding of Slimb to PAR-1-WT compared with PAR-1-T408A. Anti-Numb IP serves as a negative control. (e) In vivo ubiquitination assay showing reduced ubiquitination of PAR-1-T408A compared with PAR-1-WT. (f) Genetic interaction between PAR-1 and Slimb in the retina in each of the indicated genotypes (GMR-Gal4/ +control, n =16; GMR-Gal4>UAS-Slimb-RNAi, n =17; GMR-Gal4>UAS-PAR-1-WT, n =16; GMR-Gal4>UAS-PAR-1-WT +UAS-Slimb-RNAi, n =16; GMR-Gal4>UAS-PAR-1-T408A, n =17; and GMR-Gal4>UAS-PAR-1-T408A +UAS-Slimb-RNAi, n =17). Statistically significant differences are P<0.001 (GMR-Gal4>UAS-PAR-1-WT, GMR-Gal4>UAS-PAR-1-WT +UAS-Slimb-RNAi) as determined by Student’s t-test. Experiments were performed in triplicate. Dashed lines outline the eye contour. Values represent areas of retinal surface normalized with GMR-Gal4/ +control. Scale bar, 100 μm. (g) Western blot analysis showing non-responsiveness of PAR-1-T408 protein abundance to Slimb activity. Actin serves as a loading control. (h) Pulse-chase assay showing differential stability of PAR-1-WT and PAR-1-T408A in HEK293 cells. Actin serves as a loading control. IB, immunoblot.