Polye

The GT335 (1:500 dilution) and polyE (1:200 dilution) antibodies were obtained from AdipoGen (San Diego, CA); anti-anti-acetylated α-tubulin (1:1000 dilution) and anti-polyglutamylated tubulin B3 (1:500 dilution) were obtained from Sigma-Aldrich; anti-detyrosinated antibody was obtained from Abcam; anti-Xpress antibody (1:200 dilution) was procured from Invitrogen. Anti-RPGR antibody (1:500 dilution) was raised against an N-terminal epitope, which is common to both RPGR^const and RPGR^ORF15 isoforms. Detailed characterization of this antibody was previously reported (Ghosh et al., 2010 (link); Rao et al., 2015 (link)).

Proteins were separated using a Criterion XT precast gel (4–12% Bis-Tris, (Biorad, Hercules, CA, USA)). After electrophoresis, proteins were transferred onto a nitrocellulose membrane using the Criterion Blotter (Biorad, Hercules, CA, USA). Membranes were incubated with mouse anti-glutamate (GT335, 1:4000, Adipogen), rabbit anti-long-chain polyglutamate (polyE, 1:4,000, Adipogen), or rabbit anti-α-tubulin (EP1332Y, 1:3000, Abcam) antibodies. Immunoreactivity of proteins was visualized with Supersignal West Pico Chemiluminescence Substrate (Thermo, Rockford, IL, USA) following incubation with HRP-labeled sheep anti-mouse (1:2000, GE Healthcare Sciences, Pittsburgh, USA) or donkey anti-rabbit IgG (1:10,000, GE Healthcare Sciences) antisera.

Cell protein lysate were obtained from DE0194 and DE0193 fibroblasts using RIPA buffer (Pierce, Thermo-Fisher Scientific) (25 mM TrisHCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS), containing protease and phosphatase inhibitors (Roche diagnostic). Resolved proteins were transferred to nitrocellulose membrane (Millipore) and then probed with different primary antibodies: TTLL11 (#PA-46070, Thermo-Fisher Scientific 1/500), GT335 (Adipogen #AG-20B-0020 1/1000), PolyE (Adipogen #IN105 1/1000) and $\mathrm{\beta }$ -actin (Santa Cruz Biotechnology #sc-47778). Membranes were then incubated with horseradish peroxidase-conjugated secondary antibody for chemiluminescent substrate visualization (ECL Plus, Amersham Biosciences).