Fig. 4

The EGF-ERK axis regulates ILF3 stability and the SGOC network. a Immunoblot analysis of ILF3 protein in cells serum-starved and stimulated with EGF (100 ng/mL). b Immunoblot analysis of ILF3 protein in cells treated with EGF (100 ng/mL) and selumetinib (2 μM). c ILF3 mRNA expression in cells treated with EGF (100 ng/mL). d Immunoblot analysis of ubiquitinated ILF3 protein in DLD1 cells treated with or without EGF (100 ng/mL). MG132 (20 μM) was added to the cells 6 h before they were harvested with guanidine-HCl-containing buffer. The cell lysates were pulled down with nickel beads and immunoblotted with an anti-ILF3 antibody. e MS/MS of the ILF3 S382 phosphorylation site. The fragmentation spectrum of the typical ILF3 peptide RPMEEDGEEKS < phos > PSK (566.9075m/z, 3+) carrying the modification is shown with annotated y-ions (orange) and b-ions (green). The y-ion series covers the phosphorylation site and displays the typical neutral addition of 79.9663 Da (corresponding to the addition of PO₃). f Erk consensus phosphorylation motif of ILF3. g Immunoblot analysis of the indicated proteins from immunoprecipitates (IP) obtained from 293T cells with MG132 treatment for 6 h. h Immunoblot analysis of samples from co-IP with anti-Flag antibody in 293T cells transfected with the indicated constructs and treated with 20 µM MG-132 for 6 h. i Immunoblot analysis of ILF3 mutant in 293T cells transfected with the indicated constructs in the presence or absence of EGF. j Immunoblot analysis of samples from co-IP with anti-Flag antibody in 293T cells transfected with the indicated constructs and treated with 20 µM MG-132 for 6 h. k Immunoblot analysis of samples from in vitro kinase assay of Erk2. l Immunoblot analysis of poly-ubiquitinated ILF3 in poly-ubiquitination assays of 293T cells transfected with the indicated constructs and treated with 20 µM MG132 for 6 h. EV, empty vector. m Immunoblot analysis of the ILF3 protein turnover rate in 293T cells treated with cycloheximide (CHX). n Correlation of ILF3, and p-ERK staining in human 270 CRC tissue microarray samples. Representative images are shown.