Anti mx1antibody

Cells grown on round coverslips in 24-well plates were washed twice with PBS and fixed by the addition of ice-cold 4% paraformaldehyde for 12 min. All subsequent incubations were carried out at room temperature. Cells were permeabilized by a 15 min incubation in PBS containing 0.1% Triton X-100. Cells were treated with PBS containing 0.1 M glycine for 5 min and incubated with primary antibodies diluted in PBS–20% FCS for 30 min followed by incubation with secondary antibodies diluted 1:400 in PBS-20% FCS. Antibodies used were rat monoclonal antibody 15B6 (anti-F13) diluted 1:50, anti-Mx1antibody (Santa Cruz Biotechnology, ref SC-50509) diluted 1:200, anti-rat IgG—Alexa Fluor 594, anti-mouse IgG—Alexa Fluor 488 (Invitrogen). Finally, cells were washed extensively with PBS, mounted with FluorSave reagent (Calbiochem), and observed by fluorescence microscopy.
DNA was stained with Hoechst by incubating cell on glass coverslips with 2 mg/ml bisbenzimide (Hoechst dye. Sigma) for 30 min. To stain with To-Pro-3 (ThermoFisher ref T3605), permeabilized cells were incubated for five minutes in a 1:500 dilution of the commercial 1mM stock in PBS.

To test whether Mx1-9R fusion proteins were internalized into cells, 4 × 10⁴ cells of Madin-Darby Canine Kidney (MDCK) epithelial cells were incubated with 50 μg/mL of Mx1-9R fusion proteins for 3 or 12 h. After incubation, cells were incubated with 5 μg/mL Alexa Fluor™ 633-conjugated wheat germ agglutinin (WGA) (Thermo Fisher) for 10 min at 37 °C to stain the plasma membrane. Then, cells were washed with pre-warmed Hanks’ balanced salt solution (HBSS; Welgene) and fixed with a solution of 4% paraformaldehyde in Dulbecco’s Phosphate Buffered Saline (DPBS; Welgene), and permeabilized with 0.2% Triton-X100 in DPBS. After blocking with 1% bovine serum albumin (BSA) and 3% goat serum in DPBS, internalized Mx1 fusion proteins in MDCK cells were stained with anti-Mx1 antibody (Santa Cruz, Dallas, TX, USA) for 2 h at room temperature (RT). Cells were washed three times with DPBS for 10 min and incubated with Cy3-conjugated conjugated goat anti-mouse IgG (Jackson Immunoresearch, West Grove, PA, USA) for 45 min at RT. Cover slips were mounted using Fluoroshield mounting medium with DAPI (Abcam, Cambridge, UK), and intracellular Mx1-9R proteins were detected using an LSM800 (Carl Zeiss, Oberkochen, Germany).