The generation of sheep anti-RGS9-2 CT, sheep anti-RGS11, sheep anti-TRPM1, and sheep anti-mGluR6 antibodies has been described previously.
14–16 Rabbit anti-RGS7 (7RC1) was a generous gift from William Simonds (National Institute of Diabetes, and Digestive and Kidney Disease, National Institutes of Health, Bethesda, MD). Mouse anti-GPR179 (Ab877; Primm Biotech, Milan, Italy), mouse anti-β actin (A5441; Sigma-Aldrich, St. Louis, MO), rabbit anti-PKCα (P4334; Sigma-Aldrich), rabbit anti-CtBP2 (C10751; Assay Biotech, Sunnyvale, CA), rabbit anti-myc (A00172; GenScript, Piscataway, NJ), and rabbit anti-GST (sc-459; Santa Cruz Biotechnology, Inc., Dallas, TX) were purchased. For the detection of GPR179 by Western blotting and immunoprecipitation studies, we used the sheep anti-RGS9-2 CT antibody
15 (referred to in this study as sheep anti-GPR179 CT). This antibody was generated against a synthetic peptide with the following sequence: DSDDPRAGESGDQTTEKEVICPWESLAEGKAG. From this sequence, 14 amino acids are repeated 21 times in the C-terminus of GPR179, with a homology ranging between 36% and 72% (see
Supplementary Fig. S1A). We refer to this sequence as the conserved domain (CD). These antibodies recognize a single protein band upon Western blotting of HEK293 cells transfected with the GPR179 expression construct (see
Supplementary Fig. S1B). No immunoreactivity was observed in nontransfected HEK293 cells or in cells transfected with a GPR179 construct lacking the C-terminal domain that harbors CD sequences (see
Supplementary Fig. S1B). Furthermore, appending short CD sequences rendered an unrelated GST protein immunoreactive to sheep anti-GPR179 CT antibodies, confirming the antibody recognition epitope (see
Supplementary Fig. S1C). Consistent with a previous report that the original target of these antibodies, RGS9-2, is absent completely from the retina,
17 sheep anti-GPR179 CT antibodies recognized a single band on a Western blot with wild-type (WT) retinas (see
Supplementary Fig. S1D). In contrast, this band was shifted to a lower molecular weight region in
nob5 retinas (see
Supplementary Fig. S1D), consistent with the predicted disruption of the N-terminal portion of GPR179 via transposon recombination.
12