Mouse anti gc protein

The A549 cell monolayers were fixed (methanol, 10 min), rehydrated with PBS and permeabilized using 0.1% Triton X-100 solution for 20 min. Monolayers were incubated with primary rabbit anti-N protein (1:500; Sigma, Darmstadt, Germany) and mouse anti-Gc protein (1:50; Abcam, Waltham, MA, USA) antibodies (3 h, RT) followed by donkey anti-rabbit IgG (H + L) Alexa Fluor 647 (1:1000, Invitrogen, Rockford, USA) and donkey anti-mouse IgG (H + L) Alexa Fluor 555 (1:1000, Invitrogen, Rockford, USA), respectively. The nucleus was visualized using 4′,6-diamidino-2-phenylindol (DAPI; Sigma, Darmstadt, Germany). Images were captured using laser confocal microscope LSM 700 (Carl Zeiss) and ZEN software.

Proteins (10 µg) were loaded into wells of 12% gel, separated using electrophoresis and transferred onto polyvinylidene difluoride (PVDF) membranes (BioRad, Hercules, CA, USA). Membranes were blocked (5% non-fat dry milk, PBS + Tween 0.1% (PBS-T)) and incubated in rabbit anti-N protein (1:500, Sigma, Darmstadt, Germany) or mouse anti-Gc protein (1:500, Abcam, Waltham, MA, USA) antibodies. Secondary anti-rabbit HRP or goat anti-mouse IgG-HRP (1:2000; Sigma, Darmstadt, Germany) antibodies were used to form antibody–antibody complexes, visualized using Clarity ECL Substrate solution (BioRad, Hercules, CA, USA). Images were captured with ImageLab Software.