Cos 7 and MDCK cells were maintained in DMEM (Mediatech, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) and antibiotics at 37°C in a humidified 5% CO2 atmosphere.
Rabbit anti-LGN antibodies were described previously (Du and Macara, 2004 (link)). Rabbit anti-NuMA antibody was a kind gift from Duane Compton (Dartmouth Medical School, Hanover, NH). The following antibodies were also used: primary, monoclonal anti-GST (Santa Cruz Biotechnology, Santa Cruz, CA), anti–α-tubulin (Sigma-Aldrich, St. Louis, MO), anti-p150Glued (Cell Signaling Technology, Danvers, MA), and polyclonal anti-DYNC1H1 (Santa Cruz Biotechnology); secondary, Alexa 488–, 594–, 660–, and 680–conjugated (Invitrogen, Carlsbad, CA) and IRDye800-conjugated (Rockland Immunochemicals, Gilbertsville, PA) goat anti-mouse or rabbit antibodies. Purvalanol A was from Sigma-Aldrich, and ionomycin was from Invitrogen.
Rabbit anti-LGN antibodies were described previously (Du and Macara, 2004 (link)). Rabbit anti-NuMA antibody was a kind gift from Duane Compton (Dartmouth Medical School, Hanover, NH). The following antibodies were also used: primary, monoclonal anti-GST (Santa Cruz Biotechnology, Santa Cruz, CA), anti–α-tubulin (Sigma-Aldrich, St. Louis, MO), anti-p150Glued (Cell Signaling Technology, Danvers, MA), and polyclonal anti-DYNC1H1 (Santa Cruz Biotechnology); secondary, Alexa 488–, 594–, 660–, and 680–conjugated (Invitrogen, Carlsbad, CA) and IRDye800-conjugated (Rockland Immunochemicals, Gilbertsville, PA) goat anti-mouse or rabbit antibodies. Purvalanol A was from Sigma-Aldrich, and ionomycin was from Invitrogen.