Figure 2.

Stabilization of cell‐surface membrane type (MT)1‐matrix metalloproteinase (MMP) by growth differentiation factor 15 (GDF15). MT1‐FLAG or a control plasmid (200 ng) was co‐transfected into HEK293T cells cultured in 12‐well microplates with a control (–), GDF15 or TIMP‐2 (400 ng) plasmid, and cells were incubated for 48 h. (a) cell lysates were examined for total MT1‐FLAG expression using the anti‐MT1‐MMP monoclonal antibody 113‐5B7 (upper panel), FLAG M2 antibody (middle panel), and for the MT1‐FLAG active form using the FLAG M1 antibody (lower panel). (b) transfected cells were labeled with biotin, and immunoprecipitation was performed using anti‐FLAG M2 beads as described in Materials and Methods. Precipitated materials were blotted with IRDye^TM800‐conjugated streptavidin. Note that a 12.5‐kDa material was co‐precipitated with MT1‐FLAG from cells cotransfected with MT1‐FLAG and GDF15 plasmids. (c) MT1‐FLAG was immunoprecipitated from cells transfected with the indicated plasmid using anti‐FLAG M2 antibody beads, and co‐precipitated GDF15 was detected using western blotting with an anti‐GDF15 antibody (left panel). Aliquots of total lysates from transfected cells were examined for GDF15 expression using western blotting with the anti‐GDF15 antibody (right panel). Note that a 12.5‐kDa GDF15 mature form was predominantly co‐precipitated with MT1‐FLAG.