Fig. 4.

UV-light-induced phosphorylation of NELFE by MK2 leads to 14-3-3 binding. a Identification of p38-dependent NELFE interaction partners after UV light. SILAC-labeled U2OS cells expressing GFP-NELFE were mock-treated or irradiated with UV light. Cells were lysed and protein extracts were incubated with GFP Trap agarose. Enriched proteins were resolved on SDS-PAGE and digested in-gel into peptides. Peptides were extracted from gel and analyzed by LC-MS/MS. The scatter plot shows the logarithmized SILAC ratios of proteins quantified in the pull down. The color coding indicates the density. b NELFE interaction with 14-3-3 after UV light is p38- and MK2/3/5-dependent. U2OS cells expressing Flag-Strep-14-3-3 or an empty vector were mock-treated, irradiated with UV light or pretreated with the p38 or MK2/3/5 inhibitor, and then irradiated with UV light. Cells were lysed and protein extracts were incubated with StrepTactin sepharose. Enriched proteins were resolved by SDS-PAGE and selected proteins were detected with the indicated antibodies. c NELFE interaction with GST-14-3-3 is abolished in p38 and MK2 knockdown cells. U2OS cells were transfected with non-targeting, p38, or MK2-targeting siRNA and then irradiated with UV light. Cells were lysed and protein extracts were incubated with recombinant GST-14-3-3. Enriched proteins were resolved by SDS-PAGE and NELFE was detected using a specific antibody. d NELFE is phosphorylated after UV light on a 14-3-3-binding motif. GFP-NELFE was pulled down using GFP Trap agarose. Phosphorylation of NELFE was detected using antibodies recognizing the 14-3-3 motif. NELFE knockdown was used as control. e NELFE interacts with 14-3-3 after inhibition of P-TEFb. U2OS cells were treated with the p38 inhibitor or P-TEFb inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) and then irradiated with UV light. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3. f Dynamics of NELFE interaction with 14-3-3 after UV light. U2OS cells were exposed to UV light and left to recover for the indicated time points. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3. g NELFE interaction with 14-3-3 is partially dependent on the NER machinery. U2OS cells were transfected with a non-targeting siRNA or siRNA targeting XPC or CSB and then irradiated with UV light. After cell lysis, protein extracts were incubated with the recombinant GST-14-3-3