In vitro assessment of the effects of NF1 and SPRED1 variants on the expression and stability of NF and SPRED1
The estimated RAS GAP activity and the estimated strength of the NF-SPRED1 interaction might reflect differences in the expression and/or stability of the variant proteins. To investigate effects on NF and SPRED1 stability, we compared the wild-type and variant signals in the cell lysates by immunoblotting. In 12 cases, the mean expression of a variant was reduced to below 50% of the wild-type (Figure 4C, indicated in red). Of these, 6 also showed a significant reduction of > 50% in either RAS GAP activity, SPRED1 binding or both, and in 2 cases, p.Leu90Arg and p.Arg69_Gly96del, both RAS GAP activity and NF-SPRED1 binding were significantly reduced, but by < 50%. The remaining variants showed either significantly reduced expression (P < 0.05 Student’s paired t-test), but still > 50% of wild-type (that is, < 50 % reduction; Figure 4C, indicated in orange), or did not affect NF or SPRED1 expression or stability (P > 0.05 Student’s paired t-test; Figure 4C, indicated in black).