Fig. 5.
SHP2 regulates SOX9 abundance by modifying its phosphorylation and SUMOylation. a Diagrams showing the putative AGC family kinase phosphorylation motifs and SUMOylation sites on murine SOX9. b Images of immunostained tibial growth plate and periosteal sections, demonstrating enhanced protein SUMOylation and phosphorylation of SOX9 in 7-day-old SHP2Prrx1KO mice, compared with controls. Enlarged view of corresponding boxed areas in the periosteal regions and tibia growth plates are shown on the right (n = 3 for each genotype). c Representative fluorescence microscopic images (top) and bar graphs of geometric mean values of SOX9 and SUMO1 abundance, as determined by flow cytometric analysis (bottom), demonstrating that the abundance of SOX9 (red) and protein SUMOylation (red) were increased in SHP2-deficient OCPs and their derivatives, compared with controls (n = 3 for each genotype, *P < 0.05, Student’s t test). d Western blot analysis of total cell lysates (left and middle) and anti-SOX9 Immunoprecipitates (right) demonstrating the elevated abundance and SUMOylation of endogenous SOX9 (red arrows) in SHP2-deficient (KO) vs. -sufficient (CTR) OCPs that had been transiently transfected with pcDNA3/HA-Sumo1 plasmids. Note the increase of overall protein SUMOylation in SHP2-deficient OCPs. e Western blot analysis showing that blocking PKA activation in SHP2-deficient (cKO) chondroprogenitors with the inhibitor KT5720 compromises SOX9 phosphorylation, SUMOylation and its abundance. Images in d and e are representative of three experiments. Quantitative data relative to the controls are provided beneath each blot. TCL total cell lysate.