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. 2021 Nov 1;4:1237. doi: 10.1038/s42003-021-02744-4

Fig. 4. EGFR activation leads to MOB1 phosphorylation at Y95, 114, 117.

Fig. 4

a Immunoprecipitation of HA-MOB1 WT and 8YF. Lysates were immunoprecipitated with an antibody against HA-tag. Immunoblot of pY, HA, pEGFR (Y1068), EGFR, β-actin. EGFR-overexpressing HEK293A cells were transfected with HA-MOB1 WT or 8YF plasmid and incubated for 24 h, serum starved for 16 h, and treated with EGF (20 ng/ml) for 1 h. b Schematic of “Add-back approach”. All 8 tyrosines of MOB1 WT were mutated into phenylalanine (8YF), then each site was mutated back to tyrosine (7YF + Y). c Immunoprecipitation of HA-MOB1 WT, 8YF and 7YF + Y26, Y72, Y93, Y95, Y114, Y117, Y159, Y163. Lysates were immunoprecipitated with an antibody against HA-tag. Immunoblot of pY, HA, pEGFR (Y1068), EGFR, β-actin. EGFR-overexpressing HEK293A cells were transfected with HA-MOB1 WT, 8YF, and 7YF + Y mutant plasmid and incubated for 24 h, serum starved for 16 h, and treated with EGF (20 ng/ml) for 1 h. d The conserved amino acid sequences at Y95, Y114, Y117 of MOB1A in various species. e Immunoprecipitation of HA-MOB1 WT and 3YF. Lysates were immunoprecipitated with an antibody against HA-tag. Immunoblot of pY, HA, pEGFR (Y1068), EGFR, β-actin. EGFR-overexpressing HEK293A cells were transfected with HA-MOB1 WT or 3YF plasmid and incubated for 24 h, serum starved for 16 h, and treated with EGF (20 ng/ml) for 1 h. f Relative mRNA expression of CTGF. ANOVA with Tukey–Kramer post hoc test was used. Mean ± SEM (f); ***P <0.001; **P <0.01.