Ab78738

Reagents and chemicals were purchased from either Fisher Scientific Ltd. or Millipore Sigma unless otherwise indicated. Antibody to Stx4 (610439) was from BD Biosciences. Antibodies to MT1-MMP, GFP, SNAP23, and Munc18c (ab3644, ab78738, ab4114, and ab175238) were from Abcam. Antibodies to Munc18c and EGFR (sc-373813 and sc-03) were from Santa Cruz Biotechnology. Antibodies to β₁ integrin, β tubulin, and GAPDH (P4C10, E7-s, and DSHB-hGAPDH-2G7) were from Developmental Hybridoma Studies Bank. Antibody to FLAG (F3165) was from Millipore Sigma. All fluorescently labeled secondary antibodies and Alexa Fluor 647-conjugated phalloidin were purchased from Life Technologies. MT1-MMP inhibitors MAB3329 and NSC 405020 were purchased from Millipore Sigma and Tocris Biotechne, respectively. GFP-Stx4-FL and Stx4-N-terminal peptide was cloned as described previously (21 (link)). Stx4 1 to 15 and Stx4 15 to 29 were purchased from Invitrogen GeneArt Gene Synthesis, and subcloned into pEGFP-N1 using KpnI and HindIII restriction enzyme sites. MT1-MMP 3xFLAG and MT1-MMP T567E 3xFLAG were subcloned from pEGFP-N1, as described previously (25 (link)), into pCDNA 3.1 3xFLAG using BamHI and HindIII restriction enzyme sites.

Cells grown on glass coverslips were fixed with Histofix (Histolab) for 10 min at RT and then washed with TBS three times. Cells were blocked and permeabilized with 0.3% Triton X-100 and 1% bovine serum albumin (BSA) in TBS for 1 hour at RT and then incubated in primary antibodies to MMP14 (clone LEM-2763.1; ab78738, Abcam) and early endosome antigen-1 (clone C45B10; 3288T, Cell Signaling Technology) overnight at 4°C. After washing, coverslips were then incubated with secondary antibodies Alexa Fluor 488–conjugated goat anti-mouse (A11031, Thermo Fisher Scientific) and Alexa Fluor 568–conjugated goat anti-rabbit (A11034, Thermo Fisher Scientific) for 1 hour at RT. After washing, coverslips were mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fish Scientific). All fluorescence images were collected by laser scanning confocal microscopy (SP5-X; Leica) with Leica Application Suite software (version 2.7.3.9723), using a ×63 immersion (water) objective. Images were collected with the same settings for control and treated cells. Image analyses were performed using Fiji (50 (link)).