Figure 5.

Endogenous ISG15 induced ISGylation of ERK1 and suppressed ERK1 activity. (A) Reverse phase protein array (RPPA) analysis showed that ALST and OVCA5 showed a 65% and 45%, respectively, decrease in the levels of Tyr204 phospho-p44 ERK1 when transfected with the full-length ISG15 expression construct when compared to mock. (B) Western blot analyses demonstrated a significant decrease in the levels of Tyr204 phospho-p44 ERK and total ERK1 expression in ISG15 transfected OVCA5 ovarian cancer cells when compared to mock transfected cells. (Numbers indicate band intensities relative to the corresponding control after normalization using intensities of housekeeping gene beta tubulin). (C) Western blot analysis detected the decrease in 44kDa ERK1 protein level and the presence of ISGylated ERK1 in ISG15 transfected OVCA5 cells. (D) Western blot analysis results showed that interferon gamma treated ALST and OVCA5 ovarian cancer cells had a significant decrease in phosphorylated ERK and P38MAPK protein levels when compared to control solvent treated cells. (Numbers indicate band intensities relative to the corresponding control after normalization using intensities of housekeeping gene beta tubulin). (E) Western blot analysis results showed that interferon gamma treatment on ISG15 silenced ALST and OVCA5 ovarian cancer cells had no effect on phosphorylated ERK protein levels when compared to control solvent treated cells. (Numbers indicate band intensities relative to the corresponding control after normalization using intensities of housekeeping gene beta tubulin).