Fig. 5: c-SRC phosphorylates ACLY at Tyr682.

a A diagram illustrating the role of key enzymes (ACSS2 and ACLY) in cytosolic acetyl-CoA production during de novo fatty acid synthesis (depicted by our team using Microsoft PowerPoint software). b Enzymes involved in de novo fatty acid synthesis, including ACLY, ACC1, and FASN, were co-expressed with Flag-c-SRC in HEK-293T cells, followed by IP assay to investigate whether c-SRC interacts with ACLY, ACC1 and FASN. c HA-ACLY and Flag-c-SRC were co-transfected into U87 cells as indicated. Cells were lysed, and the lysates were subjected to anti-Flag M2 beads to enrich Flag-tagged c-SRC. The total lysate and immunoprecipitates were analyzed by Western blot. d Reciprocal immunoprecipitation was performed with anti-Flag M2 beads after transfection with HA-c-SRC and Flag-ACLY in U87 cells. e Endogenous IP assay was performed in U87 cells using an ACLY primary antibody (with rabbit IgG as negative control). f Recombinant proteins (GST, GST-ACLY, and His-c-SRC) were purified from BL21 E.coli competent cells. GST-pulldown assay was performed to determine the interaction between ACLY and c-SRC in vitro. g Recombinant GST-ACLY and His-c-SRC were purified from BL21 E.coli competent cells, followed by in vitro kinase assay to determine the direct phosphorylation of ACLY by c-SRC. h U87 cells were transfected with HA-c-SRC and Flag-ACLY and subjected to anti-Flag M2 beads for immunoprecipitation. Tyrosine phosphorylation of ACLY was detected after the precipitates were treated with or without CIAP. i HA-c-SRC and Flag-ACLY were transfected into U87 cells. After 12 h of transfection, cells were incubated with c-SRC inhibitor Saracatinib (10 μM) for another 8 h. Then IP assay was performed and tyrosine phosphorylation of ACLY was detected by WB. j Representative mass spectrum of the specific ACLY peptide with phosphorylated Tyr682 residual (red). k Flag-tagged ACLY mutants (Y854F or Y682F) were co-transfected with HA-c-SRC in HEK-293T cells. After 24 h of transfection, ACLY was immunoprecipitated and its tyrosine phosphorylation was analyzed by WB. l Alignment analysis of the residues (Tyr682, ACLY) across different species by SnapGene software (6.0.2). Experiments in (e, f, g, and k) were performed three times. Experiments in (c, d, h, and i) were repeated in 293 T cells (Supplementary Fig. 5).