Figure 5.

ORMDL3 interacts with Fis1and interaction increases after LPS stimulation.A, HeLa cells were transfected with myc-tagged ORMDL3 expressing plasmid or empty vector for 24 h and total cell lysate was immunoprecipitated (pull-down) with anti-myc antibody and IgG control and accessed for interaction of ORMDL3 and endogenous Fis1, similar result obtained for additional repeat (n = 3). B, MDM cells were transfected with myc-tagged ORMDL3 expressing plasmid and empty vector for 24 h, treated with LPS (250 ng/ml) for 6 h and total cell lysate immunoprecipitated with anti-myc antibody and IgG control and analyzed for interaction between ORMDL3 and Fis1 (n = 3). C, human MDMs were transfected with ORMDL3 proline 137 alanine mutant expressing plasmid (ORMDL3-P137A) or ORMDL3 full-length (ORMDL3) or empty vector (EV) for 24 h, treated with LPS and nigericin for 6 h and analyzed for IL-1β by ELISA (n = 6, similar result obtained for additional n = 6 donor). D, MDM were transfected with ORMDL3 proline 137 alanine mutant expressing plasmid or ORMDL3 full-length or empty vector for 24 h and analyzed for myc-ORMDL3 expression by Western blotting. Representative Western blot is shown; band intensity values normalized to GAPDH control are indicated above the band. Mean ± SEM; ∗p < 0.05; ∗∗p < 0.01; as determined by one-way ANOVA. IL, interleukin; LPS, lipopolysaccharide; MDM, monocyte-derived macrophage; ORMDL3, orosomucoid-like protein 3.