Fig. 3: RhoC interacts with LC3 via LIR motifs and undergoes autolysosomal degradation.

a Western blotting analysis of the RhoC protein levels in wild-type or RAB33A-overexpressing HeLa cells treated with 40 μg/mL CHX. These results are presented from three independent experiments. b Relative quantitation of RhoC protein levels was based on the western blotting results. (P < 0.0001). c Western blotting analysis of RhoC protein levels in HeLa cells treated with 40 μg/mL CHX, 100 nM Baf A1 and 10 μM MG132. These results are presented from three independent experiments. d Relative quantitation of RhoC protein levels was based on the western blotting results. (P < 0.0001, P = 0.8264). e, f RhoC undergoes autophagy-mediated degradation (CHX: 40 μg/mL, 6 h; Baf A1: 100 nM, 6 h; MG132: 10 μM, 6 h). HeLa cells were treated with the indicated inhibitors for 6 h, after which the RhoC protein levels were determined via western blotting. These results are presented from three independent experiments. The protein levels of RhoC were then quantified (mean ± SD. n = 3 times. two-tailed Student’s t-test). g LC3 and RhoC colocalization was visualized by immunofluorescence. Arrowheads indicate the colocalization of the two proteins. h Three predicted LIR sequences in RhoC. i, j Flag immunoprecipitates (IP) from lysates of HEK293T cells expressing V5-LC3 and Flag-tagged RhoC LIR motif mutants. These results are presented from three independent experiments. The protein levels of IP LC3-II were then quantified (mean ± SD. n = 3 times. two-tailed Student’s t-test). k, l Immunofluorescence analysis of the colocalization of LC3 and RhoC proteins with mutated LIR motifs. The colocalization of the LC3 and RhoC proteins with mutated LIR motifs was quantified (n = 10 cells). The data are presented as the mean ± SD. P values are shown; Student’s t-test.