Ab194109

The assay was conducted according to the protocols described in our previous studies^{6 (link),25 (link)}. Primary antibodies include: FZD7 (1:1000, ab64636, Abcam, MA, USA), β-catenin (1:1000, ab32572, Abcam), Cyclin D1 (1:1000, ab134175, Abcam), c-Myc (1:1000, ab32072, Abcam), and β-actin (1:1000, ab8226, Abcam). And Lamin B1 (1:5000, ab194109, Abcam) served as a nuclear internal control.

Total lysates were obtained from human articular chondrocyte cultures 48 h after transfection with empty pcDNA expression vector of pcDNA expression vector containing full length AnxA6 cDNA or from late stage human OA articular cartilage using T-PER^TM Tissue Protein Extraction Reagent from Pierce Biology Products. To extract the nuclear and plasma membrane proteins from the cell cultures we used the NE-PER^TM Nuclear and Cytoplasmic Extraction Reagent or the Mem-PER^TM Plus Membrane Protein Extraction Kit from Pierce Biology Products and followed the manufacturer’s instructions. Thirty μg of total cytoplasmic, nuclear or plasma membrane protein fractions were analyzed by SDS PAGE and immunoblotting with antibodies specific for AnxA6 (Abcam, ab7671), ß-catenin (Abcam, ab6302), Dishevelled 1 (Dvl1, Abcam, ab174679) as described previously [20 (link)]. For normalization of the protein expression levels, the membranes were immunostained with antibodies specific for beta-actin (total tissue cell lysate, Abcam, ab20272), alpha 1 sodium potassium ATPase (ATP1A1, plasma membrane fraction, Abcam, ab7671) and lamin B (nuclear fraction, Abcam, ab194109).