Fig. 5
The subcellular localization and protein expression of wild-type and four missense variants of PTPN11 were investigated in HEI-OC1 cells. (A) Immunofluorescence of HEI-OC1 cells transfected with C-terminally Myc-DDK-tagged PTPN11 wild-type, p.Glu139Asp, p.Tyr279Cys, p.Asn308Asp, and p.Gly503Arg. Cells were immunostained with anti-Myc (green) and anti-Concanavalin A (red) antibodies. The four mutants and wild-type of PTPN11 are well localized to the membrane. (B) Expression levels of pERK/ERK of PTPN11 wild-type and mutants were detected time dependently by western blotting in HEI-OC1 cells upon 30 ng/ml EGF stimulation. In comparison to the PTPN11 wild-type, the four mutants exhibited an elevation in expression level of pERK/ERK. The original immunoblots (uncropped, membrane edges visible, and standard protein size markers and expected molecular weight labeled) matched to the cropped blots (this figure) were all provided in Supplementary Fig. 5. (C) When EGF treatment was prolonged to 30 min, there was a significant difference in the pERK levels between the variant group with high penetrance (p.Glu139Asp, p.Tyr279Cys) and the variant group with low penetrance (p.Asn308Asp, and p.Gly503Arg). The band intensity was quantified by Image J. Intensity data was presented as means standard deviations from 3 independent plots.
