Anti smo

For Gpr161, Smo or Gli2 cilia localization assays, IMCD3, NIH-3T3, Smo-GFP, Smo-M2-myc, or SmoL412F-myc cells were grown to confluence on 12mm poly-L-Lysine pre-coated glass coverslips (BD Biosciences). The indicated compounds were dissolved in DMEM/3%CS and then applied to cells for 48hrs or 72hrs. Cells were either fixed in 100% methanol for 5min at −20°C or in 3.7% formaldehyde for 15min at room temperature. The cells were further permeabilized in blocking buffer (5% normal goat serum and 0.2% Triton X-100 in PBS) for 10min. Coverslips were treated with anti-Smo (provided by P. A. Beachy), anti-Myc (Cell Signaling Technology, 2272), anti-GFP (MBL International, 598), anti-Gli2 and anti-Gpr161 (provided by S. Scales, Genentech), and anti-tubulin (Sigma, T6793) in blocking buffer each for 30min. After several PBS washes, the coverslips were further incubated with Alexa Fluor 488-conjugated goat anti-rabbit, Alexa 594-conjugated goat anti-mouse, or Hoechst 33342 (Invitrogen) for 30min. 100 primary cilia were scored for the presence of Gpr161, Smo, SmoM2, SmoL412F, and Gli2 in each experiment.