Anti arl13b antibody

IMCD3 stables expressing GFP fusion proteins were plated on poly-L-lysine-coated coverslips and cilia induced by 48 hr of serum starvation. Cells were washed in PBS and fixed with 4% formaldehyde for 20 min (AcTub) or 2% formaldehyde and 50% ice-cold methanol for 15 min at 4°C (γ-Tub). Cells were permeabilized with 0.3% Triton X-100 in cytoskeletal buffer for 10 min. Cells were rinsed in 0.1% Tween 20 in PBS and blocked in 10% FBS in PBS for 30 min. For immunostaining of primary cilia, mouse 611B1 anti-acetylated α-tubulin antibody (1:5000; Sigma-Aldrich) or anti-Arl13B antibody (1:1000; Proteintech); and for basal body staining anti-γ-tubulin antibody (clone GTU-88, 1:1500; Sigma-Aldrich) and Arl3 staining anti-Arl3 antibody (1:500; Novus Biologicals) in 10% FBS in PBS were incubated overnight at 4°C. Alexa Fluor 647 or 405 anti-mouse or Alexa Fluor 647 anti-rabbit antibody (1:800; Life Technologies) was added for 45 min at room temperature. Coverslips were rinsed three times in 0.1% Tween 20 in PBS and once in PBS. Nuclei were stained with DAPI (Serva) diluted 1:10,000 in PBS for 1 min. Coverslips were fixed on glass slides with Mowiol (Merck). Images were taken using an Olympus IX81 microscope with a CCD camera and a 60× NA 1.35 objective. In all cases at least three independent staining experiments were carried out, and 100 cells were used for analysis.