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. 2013 Sep 17;1(4):e00086. doi: 10.1002/phy2.86

Figure 9.

Figure 9

Effect of castanospermine on nephrin signaling and glycosylation. 293T cells were transfected with nephrin WT, or the S366R and I171N mutants. In panel A, cells were cotransfected with an AP1 firefly luciferase reporter plasmid and renilla luciferase. Some cells were incubated with castanospermine (CST; 1 mmol/L, 18 h). (A) Lysates were assayed for luciferase activity after 48 h. Normalized luciferase activity is presented in arbitrary units. The nephrin mutants were unable to stimulate AP1 luciferase activity, compared with nephrin WT. Castanospermine did not enhance AP1 luciferase activity of the nephrin mutants. The effect of constitutively active MEKK1 transfection on AP1 luciferase activity is shown for comparison. *P < 0.0005 WT versus empty vector control (Ctrl), P < 0.0001 WT versus I171N, P < 0.0001 WT versus S366R, N = 3–6. (B and C) Cell lysates were immunoblotted with antinephrin antibody. Castanospermine induced a slight upward mobility shift in the partially glycosylated forms of nephrin WT and mutants (band 2). (C) Endoglycosidase H (Endo H) induced a complete loss of nephrin I171N (band 2) and the appearance of faster migrating bands (nonglycosylated nephrin) in both untreated and castanospermine-treated cells.