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. 2011 Jan 11;286(12):10551–10567. doi: 10.1074/jbc.M110.209759

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© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 7. — P23H opsin, glycosylation and ratio to WT opsin protein. A, retinal homogenates from PND 23 WT, P23H/+, and P23H/P23H mice were labeled with N-terminal (B6–30) and C-terminal (1D4) opsin antibodies. All lanes were loaded with 10 μg of homogenate. Opsin monomers (∼35 kDa) produced double bands due to differences in glycosylation. The molecular mass of opsin monomers shifted down to ∼30 kDa after treatment with PNGase F. Double bands were due to incomplete deglycosylation. No glycosylation difference was observed between WT and P23H/+ opsin. No detectable signal was observed in retinal homogenates from P23H/P23H mice. B, retinal homogenates from PND 28 mice were labeled with B6-30 and anti-GFP antibodies. Left panel, in homozygous GFP-tagged human opsin knock-in mice (hrhoG/hrhoG), strong bands were observed for the monomer of GFP-tagged human opsin (∼70 kDa) and its dimer (∼160 kDa). A band of human opsin with a truncated GFP tag (∼ 40 kDa) was also seen that was distinguishable from the P23H opsin band (∼35 kDa) observed in lane of P23H/hrhoG; a heterozygous hrhoG and P23H knock-in mouse. Two μg of protein were loaded. Middle panel, different amounts of retinal homogenate from P23H/hrhoG mice were loaded, from left to right: ×1, 0.175 μg; ×10, 1.75 μg; and ×100, 17.5 μg. A faint GFP-tagged human opsin monomer band was detected in lane ×1 (∼70 kDa, thick black arrow) and very faint P23H opsin monomer band was detected in lane ×10 (∼35 kDa, open arrow). The P23H band was obvious in lane ×100 (open arrow) where a fainter truncated GFP tagged human opsin monomer band was also seen (∼40 kDa, narrow arrow). Right panel, 2 μg of retinal homogenate from a P23H/hrhoG mouse labeled with anti-GFP antibody produced a single broad band at ∼70 kDa confirming the identity of GFP-tagged human opsin. C, immunoblots of 2 μg of protein samples treated with Endo H and PNGase F were resolved on 12% SDS-polyacrylamide gels (upper panels) and 10% gels (lower panels). Left panel, retinal homogenate from a WT mouse (C57BL/6J, 5-month-old) labeled with 1D4 antibody. Both WT opsin dimer and monomer were shifted to lower molecular weights after either Endo H- or PNGase F treatment. Middle panel, retinal homogenate from a P23H homozygous mouse (PND 10) labeled with 1D4 antibody evidenced a similar shift. Right panel, retinal homogenate from a P23H homozygous mouse (PND 10) labeled with B6-30 antibody. Arrowheads in upper panels indicate P23H opsin dimers resolved in 12% gels. Arrowheads in lower panels show P23H opsin dimers resolved in 10% gels. Open arrow, P23H opsin monomer. Differing from WT opsin (left), PNGase F-treated P23H opsin shifted only ∼4 kDa relative to Endo H-treated retinal homogenate. This ∼4-kDa difference arose from two N-acetylglucosamines left at the glycosylation sites. Thus, glycosylation of P23H opsin is Endo H-sensitive.