Grasp55

The proximity ligation was assay was carried out using the Sigma duolink proximity ligation assay (Sigma, DUO92101) according to the manufacturer`s instructions. Fixed HEK293T cells were permeabilized for 1h with 0.5 % Tx-100 in PBS. Afterwards cells were incubated in blocking solution for 1h at 37 °C and incubated in the primary antibody (Cx36 1:2000, Chemicon, MAB3045; Sec24B 1:1000; Cell Signaling; Grasp55 1:2000, Proteintech, 10598–1-AP) diluted in the supplied antibody diluent overnight at 4°C. At the next day coverslips were washed twice for 5 min in wash buffer A and incubated in the PLUS and MINUS PLA probes (each diluted 1:40 in dilution buffer) for 1h at 37°C. The coverslips were washed twice in wash buffer for 5 min and incubated in the ligation mix (ligase diluted 1:40 in corresponding buffer solution) for 30 min at 37°C. Afterwards coverslips were washed again in wash buffer A and incubated in the amplification mix (Polymerase diluted 1:80 in amplification buffer) for 100 min at 37 °C. Afterwards coverslips were washed 2×10 min in wash buffer B, once in 0.01x wash buffer B for 1 min and mounted with supplied mounting medium.

Protein samples were separated via SDS-PAGE in a 10 % polyacrylamide gel at 160V. Afterwards proteins were transferred onto nitrocellulose membranes using the Trans-Blot Turbo transfer system (BioRad, 1704270, 1704159). Nitrocellulose membranes were blocked at RT in blocking solution containing 5% milk powder in TBST (20mM Tris pH 7.5, 150mM NaCl, 0.2 % Tween) and incubated in the primary antibody (diluted in blocking solution, Cx36 1:500, ThermoFisher, 37–4600, RRID:AB_2533320; Tubulin 1:1000, Abnova, MAB12827; Grasp55 1:1000, Proteintech, 10598–1-AP; GFP, 1:1000, Cell Signaling Technology, 2956, RRID:AB_1196615; V5 1:1000, ThermoFisher, R960–25, RRID:AB_2556564; Syntaxin5 1:5000, Synaptic Systems, 110 053; Sec24A 1:500, ThermoFisher, PA5–66043; Sec24B 1:10000, Bethyl Laboratories, A304–876A, RRID:AB_2621071; Ergic3 1:5000, Abcam, ab129179, RRID:AB_11141068; Sec24C 1:500, ThermoFisher, PA5–59101; GOPC: 1:1000, ThermoFisher, PA5–110897, RRID:AB_2856308) overnight at 4°C on a rotating platform. At the next day blots were washed 3x with TBST for 10 min and incubated with HRP conjugated secondary antibodies (goat anti mouse HRP, 1:1000, 34130 and goat anti rabbit, 1:1000, 34160, Thermofisher) for 1h. Nitrocellulose membranes were washed 3x with TBST and incubated for 1 min in ECL substrates (Thermofisher, 32106) for chemiluminescence detection.

The following primary antibodies were used. Monoclonal antibodies against β-actin and GFP (Sigma-Aldrich); Shiga toxin B and ubiquitin (ThermoFisher); GM130, Gos28, Golgin-245, and syntaxin 6 (BD Biosciences, Franklin Lanes, NJ); α-tubulin (Developmental Studies Hybridoma Bank, University of Iowa); Arl1 (Abcam); VSV-G extracellular domain (David Sheff, University of Iowa Carver College of Medicine). Polyclonal antibodies against GCC88, Golgin-160, CI-M6PR, GRASP55, and GRASP65 (Proteintech); GCC185 (Abcam); GM130 (“N73,” from J. Seemann, University of Texas Southwestern Medical Center); TGN46 (Bio-Rad); LC3B (Cell signaling). Secondary antibodies were purchased from Jackson Laboratory (Bar Harbor, ME). Secondary antibodies used for fluorescence microscopy include fluorescence-labeled goat anti-mouse, goat anti-rabbit, and goat anti-sheep antibodies. Secondary antibodies used for Western blot include HRP-conjugated goat anti-mouse and goat anti-rabbit antibodies.