Figure 8.
Processing of MT1-MMP in HT-1080 cells. (A) Pulse-chase analysis of MT1-MMP processing. HT-1080 cells were pulsed with [35S]methionine as described and chased for 0, 15, 30, 60, and 180 min. Immunoprecipitates of cell lysates were analyzed by SDS-PAGE/autoradiography. The arrowheads mark the positions of pro, mature, and truncated forms of MT1-MMP. (B) Processing of epitope-tagged MT1-MMP in HT-1080 cells. Triton X-114 extracts of HT-1080 cells expressing MT1-MMP/FLAG (lane 2), MT1-MMP/FLAG containing the A4 mutation (lane 3), MT1-MMP/FLAG containing the AXXR mutation (lane 4), MT1-MMP containing both the A4 and AXXR mutations (lane 5), or MT1-MMP/FLAG and α1PDX (lane 6) were analyzed by immunoblotting with anti-FLAG mAb. Glycosylated proMT1-MMP could be detected when processing was blocked by the A4 or AXXR-A4 mutations in lanes 3 and 5. Extracts of HT-1080 cells transfected with a control expression vector did not reveal anti-FLAG immunoreactive products (lane 1).