Sc 166685

Retinal pigment epithelial (RPE) cells, 293T cells, and primary chondrocytes and osteoblasts were seeded onto poly L-lysine coated coverslips in 24-well plates. Confluent RPE and 293 T cells were serum-starved overnight to increase cilia lengths. All post-confluent cells were fixed in 4% PFA in PBS containing 0.1% Triton x-100 for 10 min at room temperature, then blocked in 1% Bovine Serum Albumin (Sigma, A9647) for 1 h. Primary antibodies against Arl13b (Proteintech, 17711–1-AP, 1:300), acetylated α-tubulin (Sigma T6557, 1:4000), THM2 (Custom-made Antibody, Proteintech S4132–2, 1:50), IFT81 (Proteintech, 11744–1-AP, 1:200), SMO (Santa Cruz, sc-166685, 1:50) were incubated with cells in 1% BSA overnight at 4°C. Cells were washed three times in PBS, then incubated with secondary antibody conjugated to Alexa Fluor 594 or Alexa Fluor 488 [Life Technologies, A-11005 (anti-mouse) and A-11008 (anti-rabbit), 1:500] for 1 h at room temperature. Cells were washed three times in PBS. Coverslips were inverted and mounted onto slides using DAPI Fluoromount-G (Electron Microscopy Services, 17984–24). RPE cells were imaged using a Nikon Eclipse TiE attached to an A1R-SHR confocal with an A1-DU4 detector and LU4 laser launch. All other cells were imaged using a Nikon 80i or Nikon TiS attached to a Nikon DS-Fi1 or QICAM FAST1934 camera, respectively.