Fig. 5: TKS4/5 promote ADAM-mediated shedding of MT1-MMP in acidic endosomes.

a HeLa cells were transfected with mCh-MT1-MMP. 3 h post transfection, cells were treated with metalloproteinase inhibitors as indicated. Protein lysates were made 24 h after the transfection and analysed by WB. Graph shows the quantification of the 34 kDa cleavage product of MT1-MMP. Cells treated with the lowest concentration of DMSO were set to 1. Data is from n = 3 independent experiments, mean +/− SD. One sample two-sided t-test. b MDA-MB-231 cells were transiently transfected with mCh-MT1-MMP, TKS4- or TKS5-GFP and ADAM12-myc. 24 h after transfection, cells were fixed and stained with anti-mCh, anti-GFP and anti-Myc antibodies and analysed by confocal microscopy. mCh-MT1-MMP, TKS4/5-GFP and ADAM12-myc colocalise on endosomes. Displayed is a maximum intensity projection of 2 slices à 0.9 µm section thickness. Representative of 13 (TKS4) and 6 (TKS5) confocal image acquisitions. See also Supplementary Fig. 8b for ADAM15. c Co-immunoprecipitation experiment using HeLa cells transfected with mCh-MT1-MMP, ADAM15-myc-SNAP and TKS5-GFP wt or µµ or GFP (ctrl). ADAM15-myc-SNAP preferentially co-immunoprecipitates with mCh-MT1-MMP in the presence of wt, but not mutated TKS5-GFP (“µµ”) or GFP alone. Graphs show the relative mean intensity +/−SD of the ADAM15-myc-SNAP signal quantified from cells transfected with TKS5-GFP (n = 4), TKS4-GFP (n = 3) or a combination of both (n = 4). Representative WBs for TKS4-GFP and TKS4 + 5-GFP Co-IP experiments are shown in Supplementary Fig. 8c. One-way ANOVA with Tukey’s multiple comparisons test. d MDA-MB-231-TKS5-GFP-mCh-MT1-MMP cells were treated with 25 or 50 mM NH₄Cl for 20 h. Total cell lysates were analysed by WB. Graph shows the quantification of the 34 kDa cleavage product of MT1-MMP. Data is from three independent experiments, mean +/− SD, one sample two-sided t-test.