Trafficking of Pdgfb, Gpnmb, and Angptl2 was evaluated by FRAP following previously described procedures64 . ASPS17 and null cells were transfected with with plasmids containing, DsRed-tagged Pdgfb, Gpnmb, or Angptl2. RFP-tagged actin (Invitrogen) was used as a negative control of trafficking. FRAP images were acquired with an LSM880 confocal microscope equipped with a live cell chamber (set at 37 °C) and ZEN software (Zeiss) with a 40X objective. Cells were excited with a 561 nm laser and the emission between 566 and 689 nm recorded. Images were acquired with 12 bits image depth and 512 × 512 resolution using a pixel dwell of ~1.52 μs. At least three (n ≥ 3) pre-bleaching images were collected and then the region of interest was bleached with 100% of laser power. The recovery of fluorescence was traced at every 5 min for 1 h. For drug inhibition experiments, 10 µM of Nexinhib 20 (Tocris) was added into culture media 3 h before bleaching. Fluorescence recovery rates were corrected for internal photobleaching and background, and they were normalized to pre-photobleaching intensities as previously described62 (link).
Nexinhib20
Manufactured by Bio-Techne
Sourced in United Kingdom
Nexinhib20 is a laboratory reagent produced by Bio-Techne. It functions as a specific inhibitor of the Nex1 protein. Nex1 is involved in various cellular processes. The core function of Nexinhib20 is to inhibit the activity of the Nex1 protein in experimental settings.
Automatically generated - may contain errors
Lab products found in correlation
Zen software, by Zeiss (1 mentions)
Pitstop 2, by Merck Group (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Celltracker green cmfda, by Thermo Fisher Scientific (1 mentions)
Celltracker red cmtpx, by Thermo Fisher Scientific (1 mentions)
Dynasore, by Bio-Techne (1 mentions)
Ly223982, by Cayman Chemical (1 mentions)
Mk886, by Bio-Techne (1 mentions)
Mitmab, by Bio-Techne (1 mentions)
Y 27632, by Cayman Chemical (1 mentions)
Gw4869, by Cayman Chemical (1 mentions)
12 o tetradecanoylphorbol 13 acetate tpa, by Merck Group (1 mentions)
Gm6001, by Merck Group (1 mentions)
Stem cell factor (scf), by R&D Systems (1 mentions)
Sdf 1α, by R&D Systems (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Eht1864, by Bio-Techne (1 mentions)
Mbq 167, by Selleck Chemicals (1 mentions)
Rab11a, by Abcam (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
5 protocols using nexinhib20
1
Intracellular Trafficking Dynamics Analysis
2
Multicolor Fluorescent Immune Cell Imaging
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Antibodies against mouse Itgb2 (clone M18/2; Alexa Fluor 594 conjugated), NMIIA heavy chain (clone Poly19098), and custom rhodamine-conjugated antibody to CD31 (clone 390) were obtained from BioLegend, Inc. Antibodies against CD31 (clone 390; eFluor 450 conjugated) and for quenching Alexa Fluor 488 (RRID AB_2532697) were purchased from Invitrogen. Antibodies against CD16/32 (Fc block; clone 2.4G2) and ITGB2 (clone CTB104; Alexa Fluor 488 conjugated) were obtained from BD PharMingen and Santa Cruz Biotechnology Inc., respectively. E. coli BPs (K12-strain; Texas red conjugated), CellTracker Green CMFDA, CellTracker Red CMTPX, CellMask Deep Red for PM, and rhodamine phalloidin dyes were obtained from Molecular Probes. PitStop2, monodansylcadaverine (MDC), fibrinogen (human plasma derived), and WKYMVm were purchased from MilliporeSigma. Recombinant fNLFNYK, human C5a, and human CXCL8 were obtained from Santa Cruz Biotechnology Inc., R&D Systems, and PeproTech Inc., respectively. LTB4, LY223982, Y27632, nBleb, and GW4869 were purchased from Cayman Chemical. nBleb used in this study is a photostable version of blebbistatin (Lucas-Lopez et al., 2005 ). MK886, Nexinhib20, Exo1, dynasore, and MitMAB were obtained from Tocris Bioscience.
3
Evaluation of Stem Cell Signaling Modulators
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Stem cell factor (SCF), stromal cell-derived factor 1 alpha (SDF-1α) and interleukin-3 (IL-3) were obtained from R&D systems (Minneapolis, MN). Chemical inhibitors PP3, EHT 1864, and Nexinhib20 were obtained from Tocris (Minneapolis, MN). Ascorbic acid and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were obtained from Sigma-Aldrich (St. Louis, MO). MBQ-167 was obtained from Selleckchem (Houston, TX). Recombinant fibroblast growth factor 2 (FGF-2), antibody against β-Actin and the chemical inhibitors PP2 and GM6001 were obtained from EMD Millipore (Billerica, MA). Antibodies against Rab3B, Rab3D, Rab11A and Rab27A were obtained from Abcam (Cambridge, MA). Antibodies against Caveolin-1, Rab3A, Rab5 and Rab8 were obtained from Cell Signaling Technology (Danvers, MA). An antibody against RalA was obtained from BD Biosciences (San Jose, CA). GFP-RalA and S-Ch-Cdc42 adenoviruses were generated and utilized as described previously[13 (link), 40 (link)].
4
Effects of Rab27a Inhibitor on Hair Follicle Cells
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hDPCs were cultured in Follicle Dermal Papilla Cell Growth Medium with Growth Medium Supplement Mix (PromoCell, Heidelberg, Germany) and 1% Gibco Antibiotic-Antimycotic (Thermo Fisher Scientific, Waltham, MA, USA). hORS cells were cultured in EpiLife medium with EpiLife Defined Growth Supplement (Gibco, New York, NY, USA) and 1% penicillin/streptomycin (Gibco). hDPCs and hORS cells were maintained at 37 °C in a humidified 5% CO2 incubator. We used Nexinhib20 (Rab27a inhibitor; Tocris Bioscience, Bristol, United Kingdom). For chemical treatment, hDPCs and hORS cells were seeded, and the Nexinhib20 was treated after 24 h with normal mediums, including 1% penicillin/streptomycin or Gibco Antibiotic-Antimycotic. For cell proliferation assay and migration assay, Nexinhib20 was treated to the cells with different doses (2 or 20 μM) for 3 days, and the cells were then harvested.
5
Nexinhib20 for Neural Crest Cell Culture
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Nexinhib20 (Tocris) was resuspended as a stock at 50 mM in ethanol, further diluted at 2.5 mM in ethanol or methanol (with equivalent results) and used at 2.5 µM (Johnson et al., 2016 (link)) in exosome-depleted neural crest culture medium. Diluted inhibitor or vehicle control was added to neural crest cultures after neural folds had been incubated for 3–4 h at 37°C to allow them to adhere to the fibronectin substrate.
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