Anti gm130 antibody

Cells grown on coverslips were washed gently with phosphate-buffered saline (PBS) and fixed in 4% PFA, followed by permeabilization with 0.3% Triton X-100 in PBS. The cells were then blocked for 1 h in 5% goat serum, followed by incubation overnight with primary antibodies at 4°C. Mouse anti-FLAG antibody (Beyotime biotechnology, AF519-1, 1:500 dilution), rabbit anti-FLAG antibody (Beyotime biotechnology, AF0036, 1:500 dilution), anti-GJB1 (Cx32) antibody (Santa Cruz Biotechnology,#10519, 1:150 dilution), anti-Calnexin antibody (Abcam, ab22595, 1:400 dilution), anti-GM130 antibody (Sigma-aldrich, 610822, 1:400 dilution), and anti-G3BP1 antibody (Beyotime biotechnology, AF0039, 1:150 dilution) were used. Secondary antibodies were Alexa Fluor (488, 555)-labeled goat anti-mouse and goat anti-rabbit antibodies (Thermo Fisher, 1:500 dilution). DAPI was used to visualize the nuclei. Fluorescence images were captured with a 20 × or 63 × objective on a Zeiss LSM780 laser scanning confocal microscope.

All mammalian cells were grown at 37°C with 5% CO₂. The cells tested included human HEK293T, human A549, canine MDCK, and equine NBL-6 cells. The cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Insect cells (Sf-9 and High Five) were grown in Grace’s insect media supplemented with 10% FBS at 23°C. Cells were probed for O-acetyl Sias by the use of virolectin probes and visualized by indirect immunofluorescence. Cells grown on coverslips were fixed in 4% paraformaldehyde and blocked with CarboFree (Vector Laboratories). Esterase-active treatment controls were performed by addition of active HE-Fc (at 20 μg/ml) during blocking at 37°C. Cells were permeabilized with Triton X-100. Cells were probed with HE-Fc SGRPs (at 20 μg/ml) in complex with Alexa Fluor 488-conjugated goat anti-human Fc γ-specific antibody (10:1 molar ratio) for 45 min at RT. Colocalization to the secretory cis-Golgi network was determined by utilization of an anti-GM130 antibody (Sigma). Coverslips were mounted and visualized by fluorescence microscopy using a Nikon E3000 microscope.

To generate a monoclonal antibody against ARHGAP33, a fragment of mouse ARHGAP33 was used as an immunogen (UNITECH, Kashiwa, Japan). Rabbit polyclonal anti-TrkB antibodies were generated using an extracellular domain of mouse TrkB (unpublished). The rabbit polyclonal anti-ARHGAP33 antibodies were described previously7 (link). Commercially available antibodies used were as follows: anti-ARHGAP33 (#HPA030118) antibody (Atlas Antibodies, Stockholm, Sweden), anti-GM130 (#610822) and anti-EEA1 (#9001964) antibodies (BD Transduction Laboratories, CA, USA), anti-PSD95 antibody (#MA1–046; Affinity Bioreagents, CO, USA), anti-TrkC (#3376) and anti-Tubulin (#2125) antibodies (Cell Signaling, MA, USA), anti-SORT1 (#ab16640) and anti-TrkB (#ab89925) antibodies (Abcam, Cambridge, UK), anti-SorLA (#sc136073) antibody (SantaCruz, CA, USA), anti-GM130 antibody (#G7295; Sigma, MO, USA), anti-phosphotyrosine (4G10) antibody (#05–1050; Millipore, MA, USA), anti-SORT1 antibody (#ANT-009; Alomone labs, Jerusalem, Israel), anti-Rab11 antibody (#71–5300; Invitrogen, MA, USA) and anti-Rab27 antibody (#18975; IBL, Gunma, Japan). All the antibodies were used at concentrations of 0.5–1 μg ml⁻¹.