Fig. 2: SNIP1 interacted with and was methylated at Lys301 by KMT5A.

a Immunoprecipitation followed by silver staining of the SNIP1 binding proteins. b Co-immunoprecipitation of endogenous SNIP1 with anti KMT5A antibody in BT549 (upper) and endogenous KMT5A with anti SNIP1 antibody (lower) in MDA-MB-231 cells (n = 3). c Immunoprecipitation and western blotting for Flag-KMT5A association with HA-SNIP1 protein in HEK-293T cells (n = 3). d, e GST pulldown with purified GST-KMT5A (d) and Ni-NTA pulldown with 6X His-SNIP1 (e) followed by immunoblotting with anti-His and GST antibodies (n = 3). f Immunoblot analysis of the lysine methylation of SNIP1 in control and KMT5A-knockdown MDA-MB-231 and BT549 cells. Lysates were assessed by immunoprecipitation (IP) with anti-SNIP1 and immunoblotting with anti-Lys-Methyl. g, In vivo methylation assay for the detection of SNIP1 methylation by western blotting using anti-Lys-Methyl. HA-tagged SNIP1^WT, SNIP1^K301R, SNIP1^K325R, and SNIP1^K342R mutants were immunoprecipitated with anti-HA magnetic beads from HEK-293T cells co-expressed with Flag-KMT5A (n = 3). h Autoradiography of the in vitro methylation assay using purified GST-KMT5A and 6× His-SNIP1. Top panel, 3H-SAM is the methyl donor, and methylation visualized by autoradiography. Bottom panel, total amounts of GST-KMT5A and 6× His-SNIP1 are shown by Coomassie brilliant blue (C.B.B.) staining. Empty vector (EV). Wild-type (WT) (n = 3). i Effects of wild type and methyltransferase activity-deficient mutant R336G of KMT5A on SNIP1-K301 methylation (n = 3). j Autoradiography of the in vitro methylation assay using purified GST-KMT5A^WT or methyltransferase activity-deficient mutant GST-KMT5A^R336G and 6× His-SNIP1. Top panel, 3H-SAM is the methyl donor and methylation visualized by autoradiography. Bottom panel, total amounts of GST-KMT5A^WT or -KMT5A^R336G and 6× His-SNIP1 are shown by Coomassie brilliant blue (C.B.B.) staining. Wild-type (WT) (n = 3). k In vitro methylation assay was performed with wild-type and mutant KMT5A on recombinant SNIP1 and probed with anti-K301me1 (n = 3). Data information: Panels (b–k) show 1 experiment representative of three independent experiments with similar results.