Fig. 3: UBIAD1-regulated ERK signaling initiated from the plasma membrane.

a UBIAD1 changed the localization of H-Ras under short-term (5 min) EGF treatment. HEK293T cells were transfected with plasmids as indicated. After 24 h of transfection, the cells were serum-starved for 6 h before treatment with EGF (100 ng/ml) for different periods of time, followed by confocal analysis, the same experiment was repeated three times. b UBIAD1 decreased short-term EGF-induced p-ERK. HEK293T cells were transfected with plasmids as indicated. Twenty-four hours after transfection, the cells were serum-starved for 6 h before treatment with EGF (100 ng/ml) for different periods of time. The total cell lysate was exposed to antibodies and examined by WB as indicated. The same experiment was repeated three times. c Ras aggregates in the plasma membrane after knocking down UBIAD1. HEK293T cells were transfected with sh-UBIAD1, followed by 72 h of culture and 24 h of culturing with or without 2BP, staining with pan-Ras antibody and confocal analysis. The same experiment was repeated three times. d Ras inhibitors reduced ERK phosphorylation after UBIAD1 knockdown. HEK293T cells were transfected with sh-UBIAD1, followed by 72 h of culture with or without Ras inhibitors (FTI-277, Salirasib, 2BP, tunicamycin). The total cell lysate was exposed to antibodies and WB was performed as indicated. The same experiment was repeated three times. e Melanotic masses disappeared under Ras inhibitor treatment in the heix mutant larvae. Melanotic masses were detected in long larvae following 2 weeks crosses. *p < 0.05, ***p < 0.001, Student’s t-test, (n = 3 experiments, each with 50 larvae)