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.
Ab4114
Manufactured by Abcam
Sourced in United States
Ab4114 is a laboratory reagent used for research purposes. It is a specific antibody designed to detect and bind to a particular target molecule or antigen. The core function of Ab4114 is to serve as a tool for researchers to identify and study the presence, location, or quantity of the target analyte in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 647 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Ab78738, by Abcam (1 mentions)
Ecltm western blotting detection reagent, by Bio-Rad (1 mentions)
Ab2739, by Abcam (1 mentions)
Ab96718, by Abcam (1 mentions)
Ar1178, by Boster Bio (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Df0135, by Leagene (1 mentions)
3 protocols using ab4114
1
Membrane Trafficking Regulation by Syntaxin 4
2
Western Blot Analysis of Cellular Proteins
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A2780 and SKOV3 cells were lysed in protease inhibitor (AR1178, Boster, Wuhan, China) supplemented with RIPA buffer (P0013B, Beyotime, Jiangsu, China). The western blotting of the samples was performed as described previously22 (link). Briefly, the proteins were separated by SDA-PAGE and transferred to PVDF membranes. These were overnight-incubated with primary antibodies at the appropriate concentration and 4 °C. The membranes were washed with PBST (phosphate-buffered saline Tween-20). Incubation with horseradish peroxidase-conjugated secondary antibodies was performed for 1 h at room temperature (RT). The protein bands were visualized using the ECLTM Western Blotting Detection Reagent (Bio-Rad, California, USA). The antibodies used in this study were as follows: OGT (1:1000, ab96718, Abcam, USA), O-GlcNAcylation (1:1000, ab2739, Abcam, USA), SNAP-23 (1:500, ab4114, Abcam, USA), ALIX (1:1000, ab27345, Abcam, USA), and Actin (1:1 000, bs-0061, Bioss, Beijing, China).
3
Immunofluorescent Localization of SNAP23 and VAMP3
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Cells were fixed with 4% paraformaldehyde (DF0135, Beijing leagene biotech. Co., Ltd, Beijing, China) at 25℃ for 25 min and incubation was carried out with primary antibodies at 4℃ overnight, including rabbit anti-SNAP23 (ab4114, 1: 100, Abcam) and mouse anti-VAMP3 (66488-1- Ig, 1: 100, Proteintech Group, Inc, Wuhan, China). Cells were incubated with secondary antibody at 37℃ for 1 h, including goat anti-rabbit IgG H&L-FITC (# ab6717) and goat anti-mouse IgG H&L-Cy3 (# ab6717). After incubating, cells were stained with DAPI (Beyotime) for 3 min and observed under a Nikon A1R confocal microscope.
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