Acetylated tubulin t6793

Rib cartilage or long bone cartilage was dissected out from newborn mice and digested by collagenase A (Roche, Indianapolis, IN, USA). Released cells were subsequently cultured in DMEM (Life Technology, Grand Island, NY, USA) with 10% FBS (Atlanta Biologicals, Flowery Branch, GA, USA). MEF cells were prepared and cultured according to previously described (Komatsu et al. 2011 (link)). Hedgehog conditioned medium was made by transfecting plasmid vector expressing N-terminal SHH into COS7 cells. Cultured primary chondrocytes were serum-starved in 0.5% serum for 36 hours and then treated with 100 ng/ml of SAG (Chemicon, Billerica, MA, USA) or cultured in Hedgehog conditioned medium for 4 hours. Cells were fixed in 4% PFA, permeabilized in PBS with 0.1% Triton x-100 (Sigma, St. Louis, MO, USA), incubated overnight at 4C with antibody against EVC2 (Y20, 1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), EVC (HPA008703, 1:50, Sigma, St. Louis, MO, USA), Acetylated Tubulin (T6793, 1:1000, Sigma, St. Louis, MO, USA), or Gamma tubulin (T5326, 1:1000, Sigma, St. Louis, MO). Cells were then incubated with a corresponding fluorescence secondary antibody for 1 hour at room temperature and subsequently mounted by ProLong Gold antifade mountant with DAPI (P36935, Life Technology, Grand Island, NY, USA) and observed under a confocal microscope (Nikon C1).