Gm130 ep892y

Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and blocked with 5% milk powder in PBS containing 0.1% Triton-X-100 (PBST) for 1 h, RT. Primary antibodies were applied for 1–2 h, RT or overnight, 4 °C, washed in PBST, then incubated with anti-mouse IgG or anti-rabbit IgG conjugated to horseradish peroxidase for 1 h. Following washing in PBST, membranes were visualized either using ECL (GE Healthcare) and exposed to Kodak X-Omat S film or analyzed directly on a LICOR Odyssey imager (LI-COR Biosciences, Lincoln, Ne, USA) using antibodies conjugated to fluorescent markers. Antibodies: GM-130 (EP892Y, Abcam, Cambridge, UK), GAPDH (E1C604-1, Enogene, Calnexin (SPA-860, Stressgen, San Diego, CA, USA), lamin A and C (3262, 3931), Rab1a (Santa Cruz Biotechnology, Dallas, TX, USA), Rab24 (BD bioscience, San Jose, CA, USA), gC (ab6509, Abcam, Cambridge, UK), VAPB mouse monoclonal antibody (66191-1, Proteintech, Manchester, UK), and US3 (rabbit polyclonal) and UL34 (rabbit polyclonal) antibodies were kindly provided by Dr. Thomas Mettenleiter and Dr. Barbara Klupp (Friedrich-Loeffler-Institut, Greifswald, Germany). Dr. Christopher C.J. Miller (Kings College London, London, UK) kindly provided the VAPB rabbit antibody.

The sources of purchased antibodies were as follows: FLT3 (S-18), FLT3 (SF1.340), ERK (K-23), and STAT5 (C-17) from Santa Cruz Biotechnology (Dallas, TX); FLT3 (8F2), FLT3[pY842] (10A8), FLT3[pY591] (54H1), AKT (40D4), AKT[pT308] (C31E5E), STAT5 (D2O6Y), STAT5[pY694] (D47E7), ERK1/2 (137F5), and ERK[pT202/pY204] (E10) from Cell Signaling Technology (Danvers, MA); TfR (ab84036), TGN46 (ab76282), and GM130 (EP892Y) from Abcam (Cambridge, UK); STAT5 (89) from BD Transduction Laboratories (Franklin Lakes, NJ); Calnexin (ADI-SPA-860) from Enzo (Farmingdale, NY); LAMP1 (L1418) from Sigma (St. Louis, MO) and FLT3 (MAB812) from R&D Systems (Minneapolis, MN). Horseradish peroxidase-labeled (HRP-labeled) donkey anti-mouse IgG and anti-rabbit IgG secondary antibodies were purchased from The Jackson Laboratory (Bar Harbor, MA). Alexa Fluor-conjugated (AF-conjugated) donkey secondary antibodies were obtained from Thermo Fisher Scientific (Rockford, IL). The list of antibodies with sources and conditions of immunoblotting and immunofluorescence is shown in Suppl. Table 1.

The sources of purchased antibodies were as follows: KIT (M^− 14), cathepsin D (H^− 75), STAT5 (C-17), ERK2 (K-23), ARF1 (ARFS 3F1), GBF1 (25), PTP1B (D-4), SHP-1 (D-11), and SHP-2 (B-1) from Santa Cruz Biotechnology (Dallas, TX); KIT [pY703] (D12E12), KIT (D13A2), LAMP1 (lysosome-associated membrane protein 1, D4O1S), AKT (40D4), AKT [pT308] (C31E5E), STAT5 (D2O6Y), STAT5[pY694] (D47E7), ERK1/2 (137F5) and ERK [pT202/pY204] (E10) from Cell Signaling Technology (Danvers, MA); PDI (RL90), TFR (transferrin receptor, ab84036), giantin (ab24586), and GM130 (EP892Y) from Abcam (Cambridge, UK); TFR (H68.4) from Thermo Fisher Scientific (Rockford, IL); calnexin (ADI-SPA-860) from Enzo (Farmingdale, NY); GM130 (clone 35) from BD Transduction Laboratories (Franklin Lakes, NJ); LAMP1 (L1418) from Sigma (St. Louis, MO) and KIT (104D2) from Biolegend (San Diego, CA). HRP-labeled secondary antibodies were purchased from the Jackson Laboratory (Bar Harbor, MA). Alexa Fluor-conjugated secondary antibodies were obtained from Molecular Probes (Eugene, OR).