Mouse anti acetylatedα tubulin

The serum-starved NIH3T3 cells growing on glass coverslips were fixed with 1 mL 4% paraformaldehyde (15 min), permeabilized with 2 mL 0.3% Triton X-100 (10 min), and blocked with 5% bovine serum albumin (BSA) for 1 h. After incubated with mouse anti-acetylatedα-tubulin (1:100, Abcam, USA) for 3 h at room temperature, cells were exposed to Alexa Fluor488-conjugated secondary antibody (1:200, Abcam, USA) for 1 h at 37°C. 4',6-diamidino-2-phenylindole (DAPI, Solarbio, China) was used to stain the nuclei, and photomicrographs were obtained with an inverted confocal microscope (Leica, Germany). The cells stained with acetylated tubulin antibody were considered as ciliated cells. The number of primary cilia was observed by the naked eye. Cilia length was measured using straight- or segmented-line tool for selection of fluorescent signals of ciliary marker in maximum Z intensity projected images using Image J.

Total cellular proteins (20 μg from each sample) obtained above were separated by SDS-PAGE (Solarbio, Beijing, China) under reducing conditions and transferred onto a nitrocellulose membrane (Invitrogen, Shanghai, China). Membranes were blocked in 5% dry milk and probed overnight at 4 °C with gentle rocking with anti-Runx-2 (1:1000, Abcam, Cambridge, UK), rabbit anti-BMP-2 (1:1000, Abcam), rabbit anti-COL-1 (1:1000, Abcam), mouse anti-acetylated α-tubulin (1:1000, Abcam), rabbit anti-Dynlt1 (1:800, Abcam), rabbit anti-IFT88 (1:500, Santa Cruz Biotech, Dallas, Texas), or rabbit anti-GAPDH (1:800, Bioworld, Shanghai, China). After washes, membranes were incubated with a secondary antibody (HRP-conjugated goat anti-rabbit IgG or goat anti-mouse IgG, 1:10000, Bioworld). The proteins recognized by the antibody complexes were visualized by chemiluminescence (Solarbio, Beijing, China). The optical density of each maker band was measured using Image-Pro Plus 6.0 software.