Figure 5. Cholesterol crystals via xanthine oxidase-mediated H₂O₂ production inhibits SHP2 activity.

A. Quiescent HAEC were treated with and without CC (200 μg/ml) for the indicated time periods, cell extracts were prepared and equal amounts of protein from control and each treatment were immunoprecipitated with cysteine sulfonate (CSN) antibodies and the immunocomplexes were analyzed by IB for SHP2 using its specific antibodies. The same cell extracts were also analyzed by IB for total SHP2 levels to show that the treatments do not affect its steady state levels. B & C. Quiescent HAEC were treated with and without CC (200 mg/ml) in the presence and absence of Allopurinol (50 μM), PEG-CAT (100 U/ml) or RvD1 (200 ng/ml) for 30 min, cell extracts were prepared and equal amounts of protein from control and each treatment were immunoprecipitated with CSN or SHP2 antibodies. Anti-CSN immunocomplexes were analyzed by IB for SHP2 levels using its specific antibodies and the anti-SHP2 immunocomplexes were analyzed for SHP2 activity. The bar graphs represent Mean ± SD values of three experiments. *, p < 0.05 vs control; **, p < 0.05 vs CC.