Goat anti mouse igg

Cells were lysed in Laemmli sample buffer. The lysate samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, as previously described [17 (link)]. Primary antibodies against green fluorescence protein (GFP) (Santa Cruz Biotechnology, Inc., Dallas, TX), IRF3 and phosphorylated IRF3-S396 (Cell Signaling Technology, Danvers, MA), HA (Thermo Fisher), Myc (Thermo Fisher), FLAG (Sigma-Aldrich, St. Loius, MO), GAPDH (Santa Cruz), TBK1 and phosphorylated TBK1 (Cell Signaling), IRF3 (Santa Cruz), MAVS (Santa Cruz), and β-tubulin (Sigma) were used in the blotting. Horseradish peroxidase-conjugated secondary antibodies used in this study were goat anti-rabbit or goat anti-mouse IgG (Rockland Immunochemicals). The chemiluminescence signal was collected and analyzed digitally using a ChemiDoc XRS imaging system (Bio-Rad Laboratories, Hercules, CA) and the Quantity One Program, version 4.6 (Bio-Rad).

The primary antibodies used were rabbit anti-E-cadherin, rabbit anti-N-cadherin, mouse anti-β-catenin and rabbit anti-PKD1 (1/500 for western blot and 1/50 for immunofluorescence; Santa Cruz Biotechnology, Santa Cruz, CA), goat anti-actin (1/200 for western blot; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-cyclin D1 (1/1000 for western blot, Cell signaling technology, Denvers, MA). Horseradish peroxidase-conjugated secondary antibodies used were goat anti-rabbit IgG (1/2000; Dako, Glostrup, Denmark), rabbit anti-goat IgG (1/2000 Santa Cruz Biotechnology) and goat anti-mouse IgG (1/5000; Rockland, Gilbertsville, PA). The Alexa Fluor-conjugated secondary antibodies were Alexa-Fluor-594-conjugated donkey anti-rabbit IgG, Alexa-Fluor-488-conjugated donkey anti-mouse IgG conjugated (1/200; Invitrogen, Cergy-Pontoise, France). Actin was stained with Alexa-Fluor-488-conjugated phalloidin (1/50; Invitrogen, Cergy-Pontoise, France). The nucleus was stained with 4',6-diamidino-2-phenylindole DAPI (1/50000; Invitrogen, Cergy-Pontoise, France). Gö6976 and Gö6983 were purchased from Calbiochem (Darmstadt, Germany). All other biochemicals were from Sigma-Aldrich (St. Louis, MO).

Samples were loaded on a 12.5% SDS-PAGE gel. Subsequently, proteins were transferred to nitrocellulose membranes for Western blotting. Blots were blocked with 5% skimmed milk in PBS and incubated with antibodies recognizing the influenza virus hemagglutinin tag (HA.11; Covance), GroEL2 (CCs44; J. Belisle, NIH, Bethesda, MD, USA), PGRS domain (7 (link)), or the Myc tag (dMyc; Abcam). Secondary antibodies used were horseradish peroxidase conjugated to goat anti-mouse IgG or to goat anti-rabbit (Rockland). Secondary antibodies were detected with chemiluminescence (ECL).