Mouse monoclonal anti v5 r960 25

IFN-γ-primed RAW264.7 cells were allowed to adhere to coverslips, then fixed in 4% paraformaldehyde for 15 min at 37 °C. Cells were then stained with mouse monoclonal anti-V5 (R960-25, Invitrogen dilution 1:250), polyclonal rabbit anti-gp91^phox (ab129068, dilution 1:250) and polyclonal rabbit anti-Derlin-1 (8897S, Cell Signaling Technology, dilution 1:250) diluted in staining buffer (3% BSA, 0.1% saponin, PBS). Secondary staining was performed with goat anti-rabbit IgG Alexa Fluor 488 (Invitrogen) and goat anti-mouse IgG Alexa Fluor 594 (Invitrogen). After thorough washing, cells were visualized using a laser-scanning confocal microscope (Leica SP5X) and analysed with ImageJ software (NIH).

Antibodies used were: rabbit polyclonal anti-TMEM16F (Sigma-Aldrich, HPA038958; IB 1:1000); mouse monoclonal anti-SMS2 [7D10] (Santa Cruz, sc-293384; IB 1:1000); mouse monoclonal anti-nSMase-2 (Santa Cruz, sc-166637; IB: 1:1000); mouse monoclonal anti-LAMP1 [H4A3] (Santa Cruz, sc-20011; IF 1:200); rabbit polyclonal anti-CHMP4B (Proteintech, 13683-1-AP; IF 1:300); mouse monoclonal anti-ALIX [3A9] (Biolegend, 634501, IB 1:1000); mouse monoclonal anti-Actin (Sigma-Aldrich, A1978; IF 1:1200; IB 1:10,000); rabbit monoclonal anti-Na/K-ATPase [EP1845Y] (Abcam, ab-76020; IF 1:600); mouse monoclonal anti-TSG101 [C-2] (Santa Cruz, sc-7964; IB 1:1000); mouse monoclonal anti-V5 [R960-25] (Invitrogen, r96025; IF 1:400; IB 1:1000); rabbit polyclonal anti-GFP (Novus Biologicals, NB600-303; IF 1:250); HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, 31430; IB 1:5000); HRP-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific; 31460; IB 1:5000); Cyanine Cy™2-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, 715-225-150; IF 1:400); Cyanine Cy™2-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, 711-225-152; IF 1:400); Cyanine Cy™3-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, 715-165-152; IF 1:400); and Cyanine Cy™3-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Laboratories, 715-165-150; IF 1:400).