Ab13253

Exosomes lysed with M-PER (10 μg) were separated through SDS-PAGE, then transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, United States). After blocking with bullet blocking one (Nacalai Tesque, Kyoto, Japan), the membranes were incubated with rabbit polyclonal anti-AKR1B1 (1:500, sc-33219, Santa Cruz Biotechnology, Dallas, TX, United States), goat polyclonal anti-CAPG (1:100, sc-33084, Santa Cruz Biotechnology), rabbit polyclonal anti-HSP70 antibody (1:2,000, EXOAB-HSP70A-1, System Biosciences), rabbit polyclonal anti-CD63 (1:1,000, EXOAB-CD63A-1, System Biosciences), or rabbit polyclonal anti-RAB5 (ab13253, 1:1,000, Abcam, Tokyo, Japan). Immunoreactive bands were detected using enhanced chemiluminescence (EMD Millipore, Temecula, CA, United States) after incubation with horseradish peroxidase-labeled goat anti-rabbit IgG or horse anti-goat IgG (1:5,000, Vector Laboratories, Burlingame, CA, United States). Signals were detected using C-DiGit Blot Scanner (LI-COR; Kusama et al., 2018a (link)). Total proteins were stained with colloidal gold total protein stain solution according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, United States).

RAW264.7 cells were grown in 12-well plates containing round coverslip (Thermo Fisher Scientific, Waltham, MA, USA) and infected with LCMV strains at an MOI of 5. After virus internalization, intracellular trafficking of LCMV infection was monitored in co-staining experiments using monoclonal antibody against conservative NP epitope (M104, Abcam, 1:1000 dilution, Cambridge, UK), EEA1 (monoclonal F.43.1,Thermo scientific, 1:100), Rab5 (polyclonal, ab13253, Abcam, 1:80), Rab7 (rabbit monoclonal EPR7589, Abcam, 1:100), LAMP-1 (polyclone C-20, Santa Cruz, 1:100), TLR-2 (monoclonal CD282, eBioscience, 1:100), and IRAK-1 (D51G7, Cell Signaling, 1:250). Infected cells were incubated in a CO₂ incubator for 30 to 120 min as indicated, fixed with 4% PFA, and permeabilized with 0.1% Triton X-100. After blocking of non-specific binding with 1% BSA, cells were co-stained with primary antibodies for 1 h at 25 °C before treatment with secondary IgG-TR or IgG-FITC antibodies (anti-rabbit, 1:1000 dilution for 1 h at 25 °C). Coverslips were mounted with DAPI Flu–G (Southern Biotech, Birmingham, AL, USA). Confocal images were acquired and analyzed on a Zeiss LSM 880 with a 60× oil-immersion objective at the UTMB Optical Microscopy core.

Rab5 (ab13253; Abcam) and Rab7 (PA5-52369; Invitrogen) intracellular targets were labeled in separate sets of cell cultures. MDMs were washed, fixed, blocked, and permeabilized as detailed above. Rab5 and Rab7 primary antibodies were each diluted with CD68 primary antibody (M0718; Agilent Technologies, Santa Clara, CA) in perm buffer and incubated with MDMs in the dark at room temperature for 1 hr. Following washing with perm buffer, secondary antibodies targeted to Rab5 (AF594, A11072; Invitrogen) or Rab7 (AF647, A21244; Invitrogen) along with CD68 (AF488, A11029; Invitrogen) were diluted in perm buffer and added to MDM cultures to incubate 1 hr in the dark at room temperature. MDMs were counter stained and prepared for imaging as described above.
MAP K10-GFP and M. smegmatis infected MDM cultures followed the above Rab5 and Rab7 labeling protocols with one alteration each. In the Rab5 protocol, CD68 primary antibody was coupled with an AF647 secondary antibody (115-605-205; Jackson Labs, West Grove, PA) and in the Rab7 protocol, CD68 primary antibody was coupled with an AF594 secondary antibody (115-585-205; Jackson Labs). MAP K10-GFP was detected on the 488 nm laser while M. smegmatis was also detected on the 488 nm laser after labeling with SYTO 9 diluted in perm buffer and incubated for 15 minutes in the dark. All targets were measured within CD68+ MDMs.