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3 protocols using mouse anti gm130 mab

1

Immunofluorescence and Western Blotting Protocols

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Mouse anti-Lamp1 mAb (H4A3; 1:500 for immunofluorescence or hereafter IF), mouse anti-CD63 mAb (H5C6; 1:100 for IF), and mouse anti-CD8a mAb (OKT8; 1:500 for IF) were from the Developmental Studies Hybridoma Bank. Mouse anti-GFP mAb (catalog no. sc-9996; 1:1000 for Western blotting) was purchased from Santa Cruz Biotechnology, Inc. Mouse anti-EEA1 mAb (catalog no. 610456; 1:500 for IF) and mouse anti-GM130 mAb (catalog no. 610823; 1:500 for IF) were from BD Biosciences. Horseradish peroxidase–conjugated goat anti-mouse (catalog no. 176516; 1:10,000 for Western blotting) was from Bio-Rad. Alexa Fluor–conjugated goat antibodies against mouse IgG (1:500 for IF) were from Thermo Fisher Scientific.
PNGase F was purchased from New England Biolabs. The following small molecules were commercially available: nocodazole (Merck; working concentration: 33 μm), brefeldin A (Epicenter Technologies; working concentration: 10 µg/ml), cycloheximide (Sigma–Aldrich; working concentration: 10 µg/ml), and GalNAc-O-Bn (Sigma–Aldrich; working concentration: 2 mm).
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2

Immunofluorescence Staining Protocol

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Mouse anti-EEA1 monoclonal antibody (mAb) (#610456; 1:500 for immunofluorescence or IF) and mouse anti-GM130 mAb (#610823; 1:500 for IF) were from BD Biosciences. Mouse anti-Lamp1 mAb (H4A3; 1:500 for IF) was from Developmental Studies Hybridoma Bank. Mouse anti-Myc mAb (#sc-40; 1:200 for IF), mouse anti-GAPDH mAb (#sc-47724; 1:1000 for Western blot) and mouse anti-GFP mAb (#sc-25778; 1:1000 for Western blot) were from Santa Cruz. Rabbit anti-GFP (Mahajan et al., 2019) and anti-mCherry (Sun et al., 2020) polyclonal sera were previously described. Horseradish peroxidaseconjugated goat secondary antibodies were from Bio-Rad. Alexa Fluor-conjugated goat antibodies against mouse or rabbit IgG (1:500 for IF) were from Thermo Fisher Scientific. CHX (working concentration: 10 µg/ml) and chloroquine (working concentration: 50 µM) were from Sigma Aldrich. Bafilomycin A1 (working concentration: 100 nM) was from Chemscene.
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3

Visualizing Lysosomal and Endosomal Markers

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Mouse anti-Lamp1 monoclonal antibody (mAb) (H4A3; 1:500 for immunofluorescence or hereafter IF), mouse anti-CD63 mAb (H5C6; 1:100 for IF), and mouse anti-CD8a mAb (OKT8; 1:500 for IF) were from Developmental Studies Hybridoma Bank. Mouse anti-GFP mAb (#sc-9996; 1:1000 for Western blot) was purchased from Santa Cruz. Mouse anti-EEA1 mAb (#610456; 1:500 for IF) and mouse anti-GM130 mAb (#610823; 1:500 for IF) were from BD Biosciences. Horseradish peroxidase (HRP)conjugated goat anti-mouse (#176516; 1:10,000 for Western blot) was from Bio-Rad. Alexa Fluorconjugated goat antibodies against mouse IgG (1:500 for IF) were from Thermo Fisher Scientific.
PNGase F was purchased from New England Biolab. The following small molecules were commercially available: nocodazole (Merck; working concentration: 33 µM), brefeldin A (Epicenter Technologies; working concentration: 10 µg/ml), cycloheximide (Sigma Aldrich; working concentration: 10 µg/ml), GalNAc-O-Bn (Sigma Aldrich; working concentration: 2 mM).
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