Figure 7.

Calnexin interacted with GPI-APs dependent on its N-glycan and GPI. (A) Cells were transiently transfected with WT EGFP-F–CD59, CD59 mutants (C94S and misfolded CD59), misfolded CD59 lacking GPI (C94S and G103stop*), or misfolded CD59 lacking an N-glycan (C94S and N43Q), and then they were lysed with lysis buffer containing 1% NP-40. The lysates were subjected to immunoprecipitation (IP) with anti-Flag beads. The precipitated proteins were released from the beads using Flag peptides. The input (10% of total protein) and immunoprecipitated fractions were analyzed by immunoblotting with the indicated antibodies. (B) Cells stably expressing EGFP-F–CD59 or –CD59 (C94S) were cultured. At the indicated time after protein synthesis was stopped by addition of CHX, EGFP-F–CD59 was precipitated, and coprecipitated calnexin was detected. The coprecipitated calnexin with WT CD59 at time 0 was set to 1. Relative values were calculated and are represented as means ± SEM from three independent experiments. WB, Western blot.