Expression and shedding of LDLR is similar in HepG2 WT and mutant cells.
A, SDS-PAGE Western blotting analysis of total cell extracts of HepG2 WT, SC, GALNT11 KO, and GALNT2 KO cell lines stained with anti-LDLR antibody (upper panel) and anti-calnexin (lower panel). The only immunoreactive band corresponds to the expected migration of the mature form of LDLR (molecular mass, 160 kDa). A representative experiment of at least three is shown. B, 35S-pulse-chase analysis of shed LDLR from HepG2 WT and GALNT11 KO cells using immunoprecipitation with a polyclonal anti-LDLR antibody from culture media. The signals were exposed in a PhosphorImager, and the only immunoreactive band corresponds to the expected migration of shed LDLR (molecular mass, 97 kDa). The cells were pulsed for 30 min and chased as indicated. A representative experiment of two is shown. C, flow cytometry analysis of LDLR surface expression on nonpermeabilized HepG2 WT and mutant cells as indicated. A representative experiment of at least three is shown.