Figure 3.

Stability of vector-expressed RPGR protein. The stability of AAV vector-expressed RPGR protein was analyzed by western blot or immunoprecipitation followed by liquid chromatography and mass spectrometry (LC-MS/MS). (A) RPGR protein expression in AAV-transduced Rd9 retinal tissue or pTR-RPGRco-transfected HEK293 cells was examined by western blot using anti-RPGR antibody. Alpha-tubulin was used as the loading control. Arrow heads indicate the full length of RPGR protein and the asterisk denotes the shorter form of the RPGR. (B) Whole lysate from pTR-RPGRco-transfected HEK 293 cells was immunoprecipitated by anti-RPGR antibody and separated on SDS-PAGE. After Coomassie brilliant blue R-250 staining, the bands of full-length RPGR, as shown in the dashed rectangle, were excised and pooled for LC/MS/MS analysis. (C) Amino acids in bold are the ones that have been identified by LC-MS/MS and the amino acids non-bolded were the ones that have not been covered. Although the LC-MS/MS results did not achieve 100% coverage, the intact C-terminal sequence (LKNGPSGSKKFWNNVLPHYLELK) plus the correct molecular weight assured that an intact, full-length RPGR-ORF15, without open reading frame shift, was expressed.