Gpadh

The hADSC chondrogenic microspheres and chondrocytes were lysed in radioimmunoprecipitation assay lysis buffer (Cwbio, China). The concentrations of proteins were detected using a bicinchoninic acid kit (Cwbio, China). Protein samples (25 μg) were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis, electrophoresis condition: 80 V 25 min, 120 V 60 min; then protein was transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA), transfer membrane condition: 250 mA 120 min. The membrane was blocked with 5% skim milk for 60 min. Membranes were incubated with primary antibodies specific for MMP13 (1:1000, Abcam), Col2a1 (1:1000, Abcam), SOX9 (1:1000, Abcam), GPADH (1:000, affinity), KDM6A (1:1000, Proteintech), H3 (1:1000, Proteintech) and H3K27me3 (1:1000, Proteintech). After incubation with a secondary antibody (1:5000, Cell Signaling Technology, Danvers, MA, USA), the signals were detected using a chemiluminescence kit (P90719, Millipore) and chemiluminescence system (Bio‐Rad, Hercules, CA, USA) and analysed using Image Lab Software.

Western blot, GST pull-down and co-immunoprecipitation assays were performed as previously described (Huang et al., 2015 (link)). Flag tagged human MUNC18-1, Flag tagged human syntaxin 1A (STX1A), Flag tagged human Syntaxin 3B (STX3B), and Flag tagged mouse Syntaxin 3B (Stx3B) were extracted from HeLa cells transfected with corresponding plasmids. GFP tagged human MUNC18-1 and GFP tagged worm UNC-64 were extracted from HeLa cells transfected with corresponding plasmids. GST tagged human MUNC18-1 was extracted from Escherichia coli Rosetta strain transformed with human MUNC18-1 cDNA plasmids. Mouse Syntaxin 3 (Stx3) was extracted from mouse retina lysate. The primary antibodies were as follows: MUNC18-1 (1:500, mouse monoclonal antibody, BD Biosciences, Canada), Syntaxin 3 (1:1000, rabbit polyclonal antibody, Abcam, United Kingdom), GFP (1:5000, rabbit polyclonal antibody, Proteintech, United States), FLAG (1:5000, mouse monoclonal antibody, MBL, Japan), GPADH (1:3000, mouse monoclonal antibody, Proteintech, United States), GST (1:5000, rabbit polyclonal antibody, ABclonal, United States), beta-actin (1:3000, mouse monoclonal antibody, CST, United States). Quantitative analysis of protein bands was performed by the Image J software¹.