Fig. 4
LINC00115 facilitates SETDB1 methylation of PLK3. A immunoprecipitation (IP) and IB analysis of PLK3 methylation in HEK293T cells treated with/without SETDB1 inhibitor TTD-IN (25 µM) for 12 h. B Mass spectrometry (MS) analysis showing potential methylation sites in PLK3 in BT549 and MDA-MB-231 BCSCs. A schematic representation of its amino acid sequence with all lysine (K) residues was highlighted in red. PB, Polo box domain. C LC-MS/MS spectrum of two tryptic peptides with a monomethylated residue at K106 and K200, carrying a mass of + 14.016 Da, respectively. D In vivo methylation assay of PLK3 using a pan anti-Lys-methyl antibody. Flag-tagged PLK3 WT or mutants were immunoprecipitated with anti-Flag magnetic beads from HEK293T cells co-expressed with HA-SETDB1. E Effects of methyltransferase activity-deficient mutants H1224K and C1226A of SETDB1 on PLK3 methylation in HEK293T cells. F Dot blot of PLK3-K106 or -K200 mono-methylation antibody using K106 or K200 unmodified (K106 or K200-free), and K106 or K200 monomethylated (K106 or K200-me1) peptides. G Effects of SETDB1 on PLK3 K106 and K200 methylation in MDA-MB-231/sgSETDB1 BCSCs transfected with SETDB1 sgRNA-resistant HA-SETDB1WT, SETDB1H1224K, or SETDB1C1226A mutant