Fig. 8: GRK5 bound to HDAC6 and promoted its phosphorylation.

A The colocalization of GRK5 and HDAC6 in human chondrocytes was detected by immunofluorescence double staining (400×, scale bar: 50 µm). B Co-IP was used to determine the binding of GRK5 to HDAC6 in human chondrocytes. C The human GRK5 overexpressing plasmid with different domains (with a flag tag) was constructed and co-transfected with the human HDAC6 overexpressing plasmid (with a his tag) to 293T cells. After transfection for 48 h, the binding of GRK5 and HDAC6 was detected in Co-IP. D human chondrocytes were infected with GRK5 overexpressing plasmid (with flag tag) and kinase-inactive K215R GRK5 overexpressing plasmid (with flag tag), and western blotting was used to detect flag levels. E The levels of total HDAC6 and phosphorylated HDAC6 in chondrocytes were detected by western blotting. F The levels of COL2A1 and MMP13 were detected by real-time PCR. G Chondrocyte apoptosis was tested by TUNEL staining (200×, scale bar: 100 µm; arrows represented TUNEL-positive cells). H The levels of total HDAC6 and phosphorylated HDAC6 were determined in articular cartilage tissues (n = 6) and in chondrocytes (n = 3) by co-IP and western blotting. $, p < 0.05; $$, p < 0.01. $$$, p < 0.001; $$$$, p < 0.0001. n = 3.