Figure 5.

RPN1 positively regulates FUT8 activity. A, HEK293 WT cells were treated with siRPN1 or siControl for 48 h. The cell lysates were analyzed by Western blotting with anti-FUT8 (mouse Ab), anti-RPN1, and anti-GAPDH Abs. B, the lysates of HEK293 WT cells treated with siRPN1 or siControl were incubated with the FUT8 acceptor substrate and GDP-Fuc, and the acceptor substrates and products were separated by HPLC. C, the FUT8-specific activities were calculated by the peak areas in B and shown as the mean ± S.D. (error bars) (n = 3). D, the mRNA expression levels of FUT8, MGAT3, and MGAT5 in HEK293 WT cells treated with siRPN1 were quantified by real-time PCR and shown as the values relative to those in cells treated with siControl (n = 3, mean ± S.D.). The mRNA levels were normalized to the GAPDH levels. *, p < 0.05, Mann–Whitney U test.