MTCBP-1 disrupts the interactions between MT1-MMP and actin. (A) DanG cell lysates were immunoprecipitated for actin and blotted for MT1-MMP, demonstrating an interaction between actin and MT1-MMP in PDAC cells. The asterisks indicate nonspecific bands. (B) Far-Western blot overlay indicates a direct binding between F-actin and MT1-MMP. GST and GST-ACTN-N were used as a negative control and a positive control, respectively. Actin associates with ACTN-N and MT1-MMP (arrow), but not MTCBP-1. (C) Modification of this overlay binding assay was used to test the role of MTCBP-1 in the association between MT1-MMP and F-actin. Addition of purified His-MTCBP-1 disrupts the preexisting association between F-actin and GST-MT1-CT, but not that between F-actin and GST-ACTN-N. (D–F) Actin co-sedimentation assays were performed to further define the role of MTCBP-1 in disrupting the association between MT1-MMP and F-actin. The interactions of GST or GST-MT1-CT with actin filaments was measured with an increasing amount of HIS-MTCBP-1. (D) MTCBP-1 does not affect the association between GST and F-actin. (E) Dose curve of HIS-MTCBP-1 demonstrating that HIS-MTCBP-1 displace GST-MT1-CT from the pellet to the supernatant in a dose-dependent manner from 1–5 µM. An increasing level of MT1-MMP is observed in the supernatant was observed. (F) Quantification of the co-sedimentation experiments detailed in D and E, reflecting an increase in the supernatant GST-MT1-CT levels, but not the supernatant GST levels after introducing an increasing amount of HIS-MTCBP-1. (G) Panc-1 cells were transfected with control mCherry, WT MT1-MMP-mCherry or MT1-ΔPRR-mCherry. Lysates were immunoprecipitated for actin and immunoblotted for MT1-MMP. Both WT and mutant MT1-MMP-ΔPRR bind to actin with equal intensity. Graphs shown in F and G represents normalized averages ± SEM from three independent experiments. ns, not significant. All blot measurements in kD.