SDS-PAGE and subsequent immunoblot analysis of chondrocyte extracts. Primary confluent articular chondrocytes in 60-mm Petri dishes were washed and finally set in serum-free Earl's balanced salt solution without (control, lane 1) or with glucosamine hydrochloride (50 mM, lane 2) or with Ca Iono (20 μM, lane 3) and Ca Iono with glucosamine hydrochloride (lane 4). The chondrocytes were stimulated for 4 hours. Extracts of media-cell matrix were collected as described, and 30 μl of extracts was loaded on SDS-PAGE and transblotted onto nitrocellulose membrane. Subsequently, the membranes were reacted with MDA2 monoclonal antibodies overnight and were processed. Ca Iono, calcium ionophore; Glu, glucosamine hydrochloride.