Ift81

Testes from 3–4 months old conditional Ift27 KO and control mice were pulverized on ice in lysis buffer (50 mM Tris-HCl [pH8.0], 170 mM NaCl, 5 mM ethylenediaminetetra-acetic acid, 1 mM dithiothreitol, and protease inhibitors [Complete mini; Roche Diagnostics GmbH]) containing either 1% NP-40, 0.5% sodium deoxycholate, and 0.05% SDS. After centrifuge at 1200 rpm for 10 minutes, the supernatants were collected, and protein concentrations were measured by Lowry assay. Proteins were denatured and separated under denaturing conditions by SDS-PAGE and transferred to polyvinylidene fluoride membrane. Nonspecific sites were blocked with 5% nonfat dry milk in PBST for 1 hr at room temperature, and the membranes were incubated overnight with the indicated primary antibodies (IFT25: 1:2000, Cat No: 15732-1-AP, ProteinTech; IFT74: 1:1000, Cat No: AAS27620e, from ANTIBODY VERIFY; IFT81: 1:1000, Cat No: 11744-1-AP, ProteinTech; β-actin: 1:2000, Cat No: 4967S, Cell Signaling; Antibodies against IFT20, IFT27 and IFT140 (1:2000) were from Dr. Pazour’s laboratory (Jonassen et al, 2012 (link); Keady et al, 2012 (link); Pazour et al., 2002 (link))) followed by incubation with the secondary antibodies conjugated with horseradish peroxidase (Amersham). The bound antibodies were detected with Super Signal Chemiluminescent Substrate (Pierce, Rockford, IL, USA).

Cells were seeded onto Poly-L-Lysine coated coverslips in 24-well plates. At desired experimental time points, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline containing 0.1% Triton x-100 for 10 minutes at room temperature. Cells were blocked in 2% Bovine Serum Albumin (Sigma) for 1 hour at room temperature. Cells were then incubated with primary antibody diluted in blocking solution overnight at 4°C. Primary antibodies used include: acetylated alpha-tubulin (Sigma), ARL13B (Proteintech), IFT-81 (Proteintech), γ-tubulin (Sigma), PPARγ (Cell Signaling), and CEBPα (Cell Signaling). Cells were washed and incubated with secondary antibody, AF488 or AF542 (Life Technologies) for 1 hour at room temperature. To visualize lipid droplets, slides were incubated with BODIPY (Fisher) for one hour at room temperature. Coverslips were inverted and mounted onto slides using DAPI Fluoromount-G (Electron Microscopy Services, 17984–24). Cells were visualized and imaged using a Nikon80i microscope attached to a Nikon DS-Fi1 camera.