PMC:4129401 / 15765-17398 JSONTXT

{"project":"2_test","denotations":[{"id":"25018096-17954613-2046732","span":{"begin":110,"end":112},"obj":"17954613"},{"id":"25018096-17558407-2046733","span":{"begin":782,"end":784},"obj":"17558407"}],"text":"Human TERT-immortalized retinal pigment epithelium 1 (hTERT-RPE1) cells were cultured as previously described.22 Cells were seeded on coverslips, grown to 80% confluency, and subsequently serum starved for 24 hr in medium containing only 0.2% fetal calf serum for inducing cilium growth. The cells were then cotransfected with constructs encoding mRFP-POC1B (wild-type or variants) and PalmMyr-CFP-FAM161A (wild-type) with the use of Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. Cells were fixed in 4% paraformaldehyde for 20 min, treated with 1% Triton X-100 in PBS for 5 min, and blocked in 2% BSA in PBS for 20 min. Cells were incubated with the primary antibody GT335 (cilium and basal body marker, 1:500) and α-RPGRIP1L (SNC039 + SNC040,23 transition zone marker, 1:500), diluted in 2% BSA in PBS, for 1 hr. After washing in PBS, the cells were incubated with the secondary antibody for 45 min. Secondary antibodies goat anti-mouse, goat anti-guinea pig, and goat anti-rabbit (Alexa 488, 568, and 647, respectively, 1:500; Life Technologies) were diluted in 2% BSA in PBS. Cells were washed with PBS and briefly with milliQ before being mounted in Vectashield containing DAPI (Vector Laboratories). The cellular localization of wild-type and variant POC1B proteins was analyzed with a Zeiss Axio Imager Z1 fluorescence microscope equipped with a 63× objective lens. Optical sections were generated through structured illumination by the insertion of an ApoTome slider into the illumination path and subsequent processing with AxioVision (Zeiss) and Photoshop CS4 (Adobe Systems) software."}