Figure 2.
The aromatic network at the CID–BD interface is essential for complex formation and RPGR glutamylation. (A) ITC measurement of F1140A, W1141A, and L1149A mutant miniBD peptides titrated into WT CID; n = 2 independent experimental replicates for both W1141A and F1140A mutant peptides, shown as triangles and squares; n = 3 independent experimental replicates for L1149A, shown as triangles, squares and circles. (B) Western blot of RPGR expression and glutamylation in one set of RPGR knockout mice injected with WT or mutant RPGR. Black arrow in overexposed RPGR blot indicates trace glutamylation of RPGR L1302A. Two WT mice are shown to illustrate the variability of replicate injections. Saturated pixels appear in red. Double dotted lines indicate cropping to exclude samples with failed AAV injection (uncropped Western blots, including samples with failed AAV injection provided as Source Data) LE, left eye; RE, right eye. (C) RPGR glutamylation signal normalized to RPGR expression (Glu-RPGR/RPGR, hashed bars) and RPGR levels (solid bars) for WT and mutants in AAV-injected RPGR KO mice. The RPGR expression levels are similar between the WT and mutants and higher than endogenous. Each circle or triangle indicates one retina analyzed. Circles and triangles denote different secondary antibody detection scheme (Materials and methods). Uncropped western blots including samples with failed AAV injection are provided in Source Data. Bars indicate mean ± SD. n = 7 biological replicates for RPGR WT, six for RPGR L1302A, and four for RPGR W1294C. P values from two-tailed unpaired Welch’s t test. Source data are available for this figure: SourceData F2. KO, knockout. Refer to the image caption for details. The image contains three panels labeled A, B, and C. Panel A shows an isothermal titration calorimetry graph. The x-axis represents the molar ratio, and the y-axis represents the heat of injection in calories. Data points are shown as triangles and squares for W 1141 A and F 1140 A mutants, and triangles, squares, and circles for L 1149 A mutant. Panel B displays Western blot results of R P G R expression and glutamylation in R P G R knockout mice. The blots are labeled with molecular weight markers in kilodaltons (k D a) and include annotations for R P G R and Glu-R P G R. Panel C presents a vertical bar graph showing R P G R glutamylation signal normalized to R P G R expression (Glu-R P G R slash R P G R) and R P G R levels for wild type and mutants in A A V injected R P G R K O mice. The x-axis lists the different conditions (B 6, W T, L 1302 A, W 1294 C), and the y-axis shows the relative level on a logarithmic scale. Data points are represented as circles and triangles, indicating different secondary antibody detection schemes.

The aromatic network at the CID–BD interface is essential for complex formation and RPGR glutamylation. (A) ITC measurement of F1140A, W1141A, and L1149A mutant miniBD peptides titrated into WT CID; n = 2 independent experimental replicates for both W1141A and F1140A mutant peptides, shown as triangles and squares; n = 3 independent experimental replicates for L1149A, shown as triangles, squares and circles. (B) Western blot of RPGR expression and glutamylation in one set of RPGR knockout mice injected with WT or mutant RPGR. Black arrow in overexposed RPGR blot indicates trace glutamylation of RPGR L1302A. Two WT mice are shown to illustrate the variability of replicate injections. Saturated pixels appear in red. Double dotted lines indicate cropping to exclude samples with failed AAV injection (uncropped Western blots, including samples with failed AAV injection provided as Source Data) LE, left eye; RE, right eye. (C) RPGR glutamylation signal normalized to RPGR expression (Glu-RPGR/RPGR, hashed bars) and RPGR levels (solid bars) for WT and mutants in AAV-injected RPGR KO mice. The RPGR expression levels are similar between the WT and mutants and higher than endogenous. Each circle or triangle indicates one retina analyzed. Circles and triangles denote different secondary antibody detection scheme (Materials and methods). Uncropped western blots including samples with failed AAV injection are provided in Source Data. Bars indicate mean ± SD. n = 7 biological replicates for RPGR WT, six for RPGR L1302A, and four for RPGR W1294C. P values from two-tailed unpaired Welch’s t test. Source data are available for this figure: SourceData F2. KO, knockout.

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