Hpa001593

Antibodies used in this study and their dilutions for immunocytochemistry are as follows: rabbit polyclonal anti-RPGR (Sigma #HPA001593; 1:100 dilution), mouse monoclonal anti-acetylated tubulin (Proteintech group #66200-1-Ig; 1:2500), mouse monoclonal anti-γ-tubulin (Sigma #T6557; 1:2500), rabbit polyclonal anti-γ-tubulin (Sigma #T3559; 1:2500), mouse monoclonal anti-FLAG (Sigma #F1804; 1:2000), rabbit polyclonal anti-ARL13B (Proteintech group #17711; 1:2000), rabbit monoclonal anti-GFP (Invitrogen #G10362; 1:500), rabbit polyclonal anti-PDE6D (MyBiosources #MBS7005086; 1:1000 for immunoblotting), mouse monoclonal anti-β-actin (Sigma #A1978; 1:10,000 for immunoblotting). Small interfering RNAs (siRNAs) for control (ON-TARGETplus non-targeting siRNA) and human RPGR (ON-TARGETplus SMARTpool) were obtained from GE Dharmacon.

The following antibodies were used for localization studies and Western blot: ALMS1 (1:1500–2000, rb, ab84892 Abcam; 1:500, rb, NB100-97823, Novusbio), ARL13B (1:200, ms, 73-287, Neuromab; rb, 17711-1-AP, Proteintech); TUBGCP2 (1:100, rb, 25856-1-AP, Proteintech), CEP70 (1:500, rb, ab227456, Abcam), acetylated tubulin (1:250, ms, ab24610, Abcam), CEP250 (1:200, rb, 14498-1-AP, Proteintech), Centrin-3 (1:500, rb, PA5-35865, Thermo), Centriolin (1:200, ms, sc-365521, Santa Cruz), RPGR (1:500, rb, HPA001593, Sigma Aldrich). Secondary Antibodies were Alexa488 goat anti-mouse (A11029, Molecular probes) and anti-rabbit (A11034, Molecular Probes), Alexa568 goat anti-mouse (A11031, Molecular probes) and anti-rabbit (A11036, Molecular Probes). For Western blot FLAGM2-HRP (Sigma) was used.

For the western blot, protein (30 µg) was loaded into a NuPAGE 3–8% Tris-Acetate gel (Invitrogen, Waltham, MA, USA). Gels were transferred to a 0.45 µm nitrocellulose membrane for 1.5 h at 30 V in the NuPAGE transfer buffer at room temperature. Membranes were probed overnight at 4 °C with 1:500 anti-RPGR antibody (HPA001593; Sigma-Aldrich, St. Louis, MO, USA) with the following immunogen sequence, encoded by exons 10 to 12: 379EINDTCLSVATFLPYSSLTSGNVLQRTLSARMRRRERERSPDSFSMRRTLPPIEGTLGLSACFLPNSVFPRCSERNLQESVLSEQDLMQPEEPDYLLDEMTKEAEIDNSSTVESLGETTDILNMTHIMSLN509, and they were used in previous studies for western blot [24 (link),25 (link)]. This region is common across the RPGR^Ex1–19 and RPGR^ORF15 protein isoforms. Normal human retinal protein was used as a positive control. Studies with the Sigma antibody were optimised alongside an RPGR antibody (16891-1-AP; Proteintech, Rosemont, IL, USA) provided for western blot analysis, as well as lysates from normal human retina and control human iPSCs. Vinculin (ab130007; Abcam, Cambridge, UK) was used as a loading control. The membrane was imaged using the Odyssey Fc system (LI-COR Biosciences, Lincoln, NE, USA).