Mouse anti alix antibody

All live and immunofluorescence microscopy was performed on a Nikon Eclipse Ti inverted fluorescence microscope. Image capturing was obtained using a Hamamatsu camera controller C10600 and Volocity imaging software, version 6.3 (PerkinElmer; Waltham, MA, USA). Infected cells were fixed in 3.7% paraformaldehyde (Ted Pella) for 15 m, then permeabilized with 0.1% Triton-X-100 (Fisher), blocked with 1% BSA-PBS (Fisher), and stained. Antibodies/dyes were obtained from the following sources: mouse anti-ALIX antibody from Santa Cruz biotechnology (Dallas, TX, USA), rabbit anti-VPS4 from Sigma-Aldrich (St. Louis, MO, USA), mouse anti-CEP55 from Santa Cruz Biotechnology, rabbit anti-CHMP4B from Santa Cruz Biotechnology, CellLight ER-RFP from Thermo Fisher (Waltham, MA, USA), Phalloidin 633, donkey anti-goat 488 from Invitrogen (Waltham, MA), DAPI, goat anti-mouse 488 from Thermo Fisher, anti-GFP 488, FM4-64 from Molecular Probes (Eugene, OR, USA), mouse anti-C. trachomatis LPS donated by Bob Suchland (University of Washington, WA, USA). Staining of abscission proteins: HeLa cells were seeded onto glass chamber slides and fixed with paraformaldehyde 24 h after seeding to stain for antibody localization of CHMP4B, VPS4, CEP55 and ALIX proteins. All abscission proteins are stained with antibodies labeled by GFP and nuclei is displayed by DAPI staining.

The following reagents and antibodies used in this study were purchased commercially: TNFα from AbFrontier (Seoul, South Korea); z-IETD-fmk (caspase-8 inhibitor), mouse anti-HA antibody and rabbit anti-GM130 antibody from Abcam (Cambridge, UK); 2′,7′-dichlorofluorescin diacetate (DCFH-DA), hydrogen peroxide (H₂O₂), 1-methyl-4-phenylpyridinium (MPP⁺), propidium iodide (PI), poly-D-lysine, brefeldin A (BFA), GW4869, monensin, cycloheximide (CHX), necrostatin-1 (Nec-1), DPQ, proteinase K (PK), Dynasore, heparin, heparinase III, mouse anti-β-actin, and mouse anti-Flag antibody from Sigma-Aldrich (Saint Louis, MO, USA); 4′,6-diamidino-2-phenylindole (DAPI), horseradish peroxidase (HRP)-conjugated anti-mouse antibody, and HRP-conjugated anti-rabbit antibody from Thermo Fisher Scientific, Inc. (Rockford, IL, USA); mouse anti-Flotillin-1 from BD Biosciences (San Jose, CA, USA); bafilomycin A1, mouse anti-Alix antibody, mouse anti-β-Galactosidase antibody (40-1a), rabbit anti-Calregulin antibody, mouse anti-CD63 antibody, mouse anti-Parkin antibody, and mouse anti-FAF1 antibody from Santa Cruz Biotechnology (Dallas, TX, USA); mouse anti-Hsc70 antibody and mouse anti-Hsp90 antibody from Enzo Life Sciences (Farmingdale, NY, USA); And zVAD-fmk (z-VAD) from Calbiochem (Darmstadt, Germany).