Fig. 4: LEMD3 interacts with CBX3.
a Schematic diagram of the LEMD3 interactome. b Coimmunoprecipitation (Co-IP) assays of HEK293T cells cotransfected with LEMD3-FLAG and HA-CBX3 plasmids for 48 h. The lysates were immunoprecipitated with a control IgG antibody, anti-FLAG antibody, or anti-CBX3 antibody, followed by immunoblotting with the indicated antibodies. c Co-IP assays of endogenous LEMD3 and CBX3 in rat VSMCs. The lysates were immunoprecipitated with a control IgG antibody, anti-LEMD3 antibody, or anti-CBX3 antibody, followed by immunoblotting with the indicated antibodies. d Schematic illustration of LEMD3 domain depletion mutations used to evaluate the interaction with CBX3. The presence or absence of the deletion mutant binding to CBX3 was defined as + or −, respectively. e HEK293T cells were cotransfected with the CBX3 plasmid and the full-length LEMD3-FLAG, ΔC-terminal-FLAG, or ΔRRM-FLAG plasmid for 48 h. The cell lysates were immunoprecipitated with an anti-CBX3 antibody, and the precipitates were analyzed by immunoblotting with an anti-FLAG antibody. f Coimmunoprecipitation (Co-IP) assays of HEK293T cells cotransfected with RRM-FLAG and HA-CBX3 plasmids for 48 h. The lysates were immunoprecipitated with a control IgG antibody, or anti-FLAG antibody, followed by immunoblotting with the indicated antibodies. g, h Representative western blotting and quantification of VSMC contractile marker expression in the A7r5 rat VSMC cell line transfected with the scrambled siRNA (20 nM) or siRNA targeting Lemd3 (20 nM) for 12 h, followed by transfection with the pcDNA3.1, LEMD3-FLAG, or ΔRRM-FLAG plasmid for 48 h. GAPDH was used as an internal control. The data are presented as the relative fold changes compared with that of the pcDNA3.1+vehicle group (n = 5 independent experiments). i, j Representative western blotting and quantification of VSMC contractile marker expression in the A7r5 rat VSMC cell line transfected with the pcDNA3.1 or LEMD3-FLAG plasmid for 12 h, followed by transfection with the scrambled siRNA (20 nM) or siRNA targeting Cbx3 (20 nM) for 48 h. GAPDH was used as an internal control. The data are presented as the relative fold changes compared with that of the pcDNA3.1+siRNAscramble group (n = 3 independent experiments). Data shown in (b, c, e and f) are from one representative of three independent experiments with similar results. Error bars indicate s.e.m. Statistical analysis was performed using two-way ANOVA followed by Bonferroni’s multiple comparisons test for (h) and (j).
