A Role for IFITM Proteins in Restriction of Mycobacterium tuberculosis Infection

Ranjbar, Shahin; Haridas, Viraga; Jasenosky, Luke D.; Falvo, James V.; Goldfeld, Anne E.

A549 cells stably transduced with the IFITM3-V5 tag construct or the empty vector were seeded on a small coverslip in 12-well plates in fresh media and were infected with MTb-mCherry for 3 hr, after which the cultures were washed and fresh media were added. Cells were further incubated at 37°C for 20 hr. MTb-infected cells were fixed with 4% paraformaldehyde before they were blocked and permeabilized with buffer containing 5% normal donkey serum and 0.3% Triton X-100 in PBS for 2 hr. Cells were labeled with 1:150 rabbit anti-Rab5 or 1:100 rabbit anti-Rab7 antibody (Cell Signaling Technology) overnight in antibody dilution buffer containing 1% BSA followed by 1:500 secondary donkey antirabbit DyLight 649 antibody (BioLegend) for 2 hr. Cells were further labeled with mouse fluorescein isothiocyanate (FITC) anti-V5 antibody (Invitrogen) overnight, after which slides were mounted in mounting media containing DAPI. Images were captured with an Olympus (FV1000) confocal microscope and Fluoview Software 2. Analysis was performed using ImageJ software.