Western blot analysis of GPA after hydrolysis of N-glycan chains.

(A) Following hydrolysis of N-glycan chains by PNGase F, red cell membrane proteins (2 μg/lane) were separated on a 10% to 20% tricine gradient gel and then immunoblotted with BRIC 163 antibody. (B)GYPA transgenic mouse red cell proteins treated with PNGase F were solubilized with increased concentrations of SDS, incubated for 3 minutes at either 25°C or 100°C, separated on a 10% to 20% tricine gradient gel, and then immunoblotted with BRIC 163 antibody. (−) and (+) refer to proteins before and after enzyme treatment, respectively. MW indicates molecular weight.