Mouse monoclonal anti gm130 antibody

The following antibodies were used: mouse monoclonal anti-β-actin (cat. no. A1978, 1:10,000; Sigma-Aldrich), rabbit polyclonal anti-FLAG (cat. no. 2368, 1:1000; Cell Signaling), mouse monoclonal anti-PARP-1 (sc8007, 1:1000; Santa Cruz), rabbit polyclonal anti-calnexin (sc11397, 1:1000; Santa Cruz), mouse monoclonal anti-V5 antibody (R960-25, 1:4000; Invitrogen), rat monoclonal anti-HA antibody (12158167001, 1:4000; Roche), rabbit polyclonal anti-HA antibody (71-5500, 1:1000 Invitrogen), goat polyclonal anti-calnexin (sc-6465, 1:200; Santa Cruz), sheep anti-TGN46 (AHP500, 1:200 AbD Serotec) and mouse monoclonal anti-GM130 antibody (610823, 1:200; BD Biosciences). Goat anti-mouse (cat. no. 31430, 1:5,000) and goat anti-rabbit IgG conjugated to horseradish peroxidase (31460, 1:5,000) were from Thermo Fischer Scientific. The following secondary antibodies were used for indirect immuno-fluorescence: FITC-conjugated donkey anti-sheep/goat (STAR88F, 1:200; AbD Serotec), Cy3-conjugated donkey anti-mouse IgG (715-165-150, 1:200; Jackson ImmunoResearch), Cy5-conjugated donkey anti-rabbit IgG (711-175-152, 1:200; Jackson ImmunoResearch), Cy2-conjugated donkey anti-mouse IgG (715-225-150, 1:200; Jackson ImmunoResearch), Cy5-conjugated donkey anti-goat IgG (705-175-147, 1:200; Jackson ImmunoResearch), Cy3-conjugated donkey anti-rabbit IgG (711-165-152, 1:200; Jackson ImmunoResearch).

Cells (5–8 × 10⁴) were seeded on fibronectin-coated coverslips in 24-well plates and maintained with complete DMEM medium at 37 °C. After 24 h, COS-7 transfected cells were incubated with HaloTag TMR Direct Ligand (Promega) at a final concentration of 1 μM. The Golgi area was immunostained with the mouse monoclonal anti-GM130 antibody (BD Bioscences, 6108223) and Alexa Fluor 488 goat anti-mouse secondary antibody (Life Technologies, A11017). Samples were analysed with an Olympus FV300 laser-scanning confocal microscope equipped with a blue argon (488 nm) laser, a green helium–neon (543 nm) laser, and FluoView 300 software (Olympus Biosystems). Cells were imaged with a 60 × oil-immersion objective (1.4 NA). Z-stack images of optical sections were digitally recorded, 3D reconstructed with ImageJ software and the cell surface area was measured for each cell. For each experimental point, at least 120 cis-Golgi acquisitions were analyzed and three technical replicas were performed. The results are shown graphically on an arbitrary scale (au).

PcDNA SH-SY5Y, TrkA SH-SY5Y, and TrkAIII SH-SY5Y cell lines were grown in recommended medium (RPMI or Dulbecco's modified Eagle's medium) supplemented with 10% foetal calf serum and appropriate antibiotics (phleomycin [Zeocin], penicillin, and streptomycin), as described previously [1 (link)-4 ]. Cell culture reagents, MG-132, geldanamycin, cytochalasin D, nocodozol, proteinase inhibitor Cocktail, Bis-benzimide tri-hydrochloride, propidium iodide, Histodenz^TM, NGF, K252a, Go6976 and GW441756 were purchased from Sigma-Aldrich (St. Louis MO). Endoglycosidase H was from Roche Italia (Milan, It). The pan Trk inhibitor CEP-701 was from Cephalon Inc. (West Chester, PA). Polyclonal anti-TrkA (C14) and monoclonal anti-Phospho-tyrosine (pY99) antibodies were from SantaCruz (Santa Cruz, Ca). The polyclonal anti-α-tubulin antibody was from Sigma (St. Louis, MO). Polyclonal anti-phospho-Y674/675 TrkA, anti-COPI, COPII and mouse monoclonal anti-Calnexin and anti-TGN46 antibodies were from Abcam (Cambridge, UK). The polyclonal anti-phospho-Y490 TrkA antibody was from Cell signalling (Danvers, MA). Mouse monoclonal anti-GM130 antibody was from BD Bioscience (San Jose, Ca). FITC and TRIC-conjugated secondary anti mouse and anti-rabbit IgG antibodies were from Jackson Immune Research (Bar Harbor, Maine). VectorMount^TM medium was from Vector Laboratories (Berlingame, Ca).

HeLa cells were obtained from the American Type Culture Collection and were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Nacalai Tesque, #08458-45) containing 10% fetal bovine serum (Equitech Bio, #268-1). Rabbit polyclonal anti-TOM20 antibody was purchased from Santa Cruz Biotechnology (#sc-11415), and mouse monoclonal anti-GM130 antibody was from BD Biosciences (#610822). Alexa Fluor 555 conjugated anti-mouse IgG (#A31570), Alexa Fluor 555 conjugated anti-rabbit IgG (#A31572), Alexa Fluor 555 conjugated streptavidin (#S21381), Alexa Fluor 647 conjugated streptavidin (#S21374), and Alexa Fluor 647 conjugated wheat germ agglutinin (WGA) (#W32466) were from Thermo Fisher Scientific. Biotinylated WGA (#B-1025) was from Vector Laboratories. DyLight 549 (#S000-42) and DyLight 649 (#S000-43) conjugated streptavidins were from Rockland. HiLyte Fluor 555 (#AS-60666) and HiLyte Fluor 647 (#AS-60667) conjugated streptavidins were from Anaspec. iFluor 555 (#16989) and iFluor 647 (#16996) conjugated streptavidins were from AAT Bioquest. ATTO 647 (#AD647-61) streptavidin was from ATTO-TEC. SPICA dye-conjugated streptavidin was from Fujifilm Wako Chemicals. Biotin-SP AffiniPure goat anti-rabbit IgG (#111-065-144) was purchased from Jackson ImmunoResearch.