Fig. 2: p38 binds and phosphorylates ACSL4 in vitro.

a The proteins that bind with p38 were identified by IP/MS analysis. Created by Adobe Illustrator. b p38 binds to ACSL4. Beads coated with p38-3 × Flag fusion were incubated with cell lysate. Endogenous ACSL4 was detected by Western blot. c Modeling of p38 binding with ACSL4. ACSL4 is colored brown, and p38 is colored gray. d p38 and ACSL4 bind with each other exogenously. Flag-ACSL4, V5-p38 plasmids were co-transfected into HEK293T cells. Proteins were extracted after 36 h transfection. The V5- or Flag-tagged proteins were immunoprecipitated with anti-V5 (left) or anti-Flag (right), and then Western blot was performed. e p38 and ACSL4 bind with each other endogenously. Proteins extracted from KYSE150 were immunoprecipitated by anti-ACSL4 (left) or anti-p38 (right), and immunoprecipitated complexes were detected using the indicated antibody. f BiFC Assay was used to visualize p38-ACSL4 interaction in vivo. Vc-ACSL4, Vn-p38 and vector plasmids were co-transfected into HEK293T cells. After 36 h transfection, green fluorescence was observed. Results are shown as mean ± SD, n = 3 biological replicates. P values were determined using one-way ANOVA. g The phosphorylated site of ACSL4 by p38 was predicted using NetPhos 2.0 web server. h p38 kinase phosphorylates ACSL4 at T679 in vitro. Active p38 protein was incubated with ACSL4-GST fusion protein or simultaneously treatment with 10 μM SB202190. i Active p38 protein was incubated with ACSL4-3 × Flag fusion protein or harboring ACSL4-T679A-3 × Flag mutation protein in kinase reaction buffer and then checked by Western blot using antibodies as indicated. j Phosphorylation of ACSL4 at Thr679 was essential for its enzyme activity and dimerization. HEK293T cells were co-transfected with Flag-ACSL4 and HA-ACSL4 plasmids or with Flag-ACSL4-T679A and HA-ACSL4-T679A, Flag-ACSL4-T679D and HA-ACSL4-T679D plasmids. The protein was prepared, and Flag-ACSL4 was immunoprecipitated, followed by Western blot analysis using an anti-HA antibody. Representative immunoblots shown in figures were repeated three times independently with similar results. Source data are provided as a Source Data file.