Sc 5571

LC3 punctate and LAMP2 staining and quantification were performed as described previously^{12 (link)}. After treatment, cells on coverslips were washed twice with PBS, fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.25% Triton X-100 in PBS for 10 min, and immunostained with primary Abs (anti-LC3; PM036, MBL International, 1:400) or (anti-LAMP2; sc-5571, Santa Cruz, 1:400) for 2 h. Cells were washed to remove primary Abs and incubated with the fluorescently labeled secondary Ab (Alexa 488-conjugated anti-rabbit IgG; A17041, Molecular Probes, 1:400) for 2 h. Nuclei were stained by incubation with DAPI (D9564, Sigma) for 5 min. After mounting, fluorescence images were acquired using a confocal laser-scanning microscope (LSM 710, Zeiss, Thomwood, NY, USA).
LC3 punctate dot fluorescence intensity was measured using ImageJ analysis software. For colocalization analysis, the co-distribution of LC3 autophagosomes or LAMP2 phagolysosomes with bacterial phagosomes were quantified using the ImageJ analysis software. Each condition was assayed in triplicate, and at least 100 cells per well were counted.

DOPC and DOPS were purchased from Avanti Polar Lipids (Alabaster, AL, USA). ATP and ascorbic acid were purchased from Sigma-Aldrich, CHAPS from Anatrace (Maumee, OH, USA), ProteaseARREST protease inhibitor cocktail from G-Biosciences (St. Louis, MO, USA) and 1D4 peptide from Biomatik (Kitchener, ON, USA). MG132 was from ApexBio (Houston, TX, USA). HEK293T and HeLa cells were obtained from American Type Culture Collection through Cedarlane Laboratories (Burlington, Ontario, Canada). The Rho1D4 antibody initially produced in house (Hodges et al., 1988 (link); Molday and Molday, 2014 (link)) was obtained from the University of British Columbia (https://uilo.ubc.ca/industry-partners/access-ubc-technologies). The primary antibody against tubulin was from Abcam (ab15568; Toronto, Ontario, Canada) and the antibody against LAMP2 was from Santa Cruz Biotechnology (sc-5571; Dallas, TX, USA). Primary antibodies against ATP8A2 (ATP6C11 and Cdc50-7F4) have been described previously (Coleman et al., 2009 (link); Coleman and Molday, 2011 (link)). Restriction enzymes, T4 DNA ligase, Antarctic Phosphatase and Phusion polymerase were acquired from New England Biolabs (Whitby, Ontario, Canada). Primers used for mutagenesis were ordered from Integrated DNA Technologies (Coralville, IA, USA).