Fig. 1: Src directly interacts with and phosphorylates lipin-1 upon mitogenic stimulation.

a Interaction between endogenous lipin-1 and Src. Src in wild-type (WT) and LPIN1-knockout (KO) MDA-MB-231 cells were immunoprecipitated (IP), followed by immunoblotting with antibodies indicated. TCL, total cell lysate. b GST pull-down assay was performed by using bacterially purified His-lipin-1 and GST or GST-Src, followed by immunoblotting. c Lipin-1 was phosphorylated by Src in vitro. Bacterially expressed His-lipin-1 was incubated with His-WT-Src or His-KD-Src (a kinase-dead form of Src) in a kinase assay buffer, followed by immunoblotting. d Lipin-1 tyrosine phosphorylation stimulated by serum, or/and EGF. MDA-MB-231 cells were maintained in a serum-free medium for 4 h, and then treated with or without EGF, serum or serum plus EGF for additional 30 min. Cell lysates were analysed by immunoblotting with antibodies indicated. e Increased Src-dependent tyrosine phosphorylation of lipin-1 after EGF treatment. MDA-MB-231 cells expressing shRNA against SRC or Renilla as a control were maintained in a serum-free medium for 4 h, followed by stimulation with or without EGF for 30 min. Lipin-1 was immunoprecipitated, followed by immunoblotting. Quantification of the ratio of phosphorylated lipin-1 to total lipin-1 is displayed as a scatter plot. P-Tyr-lipin-1, phosphorylation of lipin-1 on tyrosine. f The tyrosine phosphorylation of lipin-1 in SRC-KO MDA-MB-231 cells reconstituted with Src. After 30 min of EGF incubation, SRC-KO MDA-MB-231 cells stably expressing Flag-tagged WT-Src or KD-Src were lysed and subjected to IP with anti-lipin-1 antibody. Quantification of the ratio of phosphorylated lipin-1 to total lipin-1 is displayed as a scatter plot. e, f were quantified in each biological replicate (n = 3); data are mean ± s.e.m.; ordinary two-way ANOVA, followed by Sidak in e, f; ***P < 0.001, N.S. not significant. Source data are provided as a Source Data file.