RAGE(C259S/C301S) is unstable and deglycosylated.

<p>(<b>A</b>) Cyclohaximide chase and IB to compare the protein decay of RAGE(WT) and RAGE(C259S/C301S) in the cells. For FLAG-RAGE transfected cells, 5 µg of lysates were used; whereas for FLAG-RAGE(C259S/C301S) transfceted cells, 10 µg of lysates were used due to the lower expression. After IB with anti-FLAG antibodies, the blot was striped, and reprobed with anti-β-actin antibodies as a loading control. CHX: cyclohaximide. (<b>B</b>) Intracellular decay rate of RAGE and RAGE(C259S/C301S) calculated from two CHX chase experiments. The blot intensity was measured with a Kodak Gel Logic 2200 Imaging System and processed with molecular imaging software. The starting point was used as 100% and blot intensity from each time point was calculated relative to the 0 time point. The intensity value of each point was expressed as mean ± SEM, and d_1/2 was calculated when 50% of the protein I decayed. <i>C</i>, RAGE cysteine-to-serine mutants are deglycosylated. Cell lysates from FLAG-tagged RAGE and RAGE cysteine-to-serine mutants were treated with PNGase F, as described, and resolved on a SDS 4–12% NuPAGE gel.