Fig. 5. MT1-MMP processing takes place in an intracellular, post-Golgi compartment. (A) MT1-MMP-transfected A375 cells were pulse-labeled with [³⁵S]methionine for 5 minutes. Labeled proteins were accumulated in the trans-Golgi network by incubating the cells at 20°C for 30 minutes, then released at 37°C for different lengths of time, as indicated. Triton X-100-soluble (S) and insoluble (P) fractions were separated by ultracentrifugation, immunoprecipitated and analyzed by SDS-PAGE/autoradiography. Tannic acid 0.5% was added during the 20°C block, 10 minutes prior to release at 37°C for 60 minutes. (B) Tannic acid completely blocks arrival of proteins to plasma membrane. A375 cells were transfected with the ts045 temperature sensitive mutant of VSV-G-GFP chimera, incubated at 40°C overnight, blocked at 20°C for 30 minutes then chased at 32°C for the indicated time in the absence or presence of 0.5% tannic acid. Panels illustrate representative control (top) and treated (bottom) cells. VSV-G staining on PM detected from non permeabilized cells with an antibody raised against its extracellular domain is shown in red. Bar, 10 µm. The graph shows ratio of VSV-G on plasma membrane to total VSV-G at indicated chase time. (C) EGTA treatment: after incubating the cells at 20°C for 30 minutes, the MT1-MMP-transfected pulsed A375 cells were treated (lane 3) or not (lane 2) with 5 mM EGTA for 30 minutes at 37°C. Only the soluble fraction is shown. MT1-MMP molecular forms in lanes 2 and 3 were quantified with the public domain ImageJ v.1.3 software and plotted on the reported chart as a ratio of MT1-MMP processing.