NOMO relies on the predicted Ig 1/10/11 interface for functionality. (A) Spinning disc confocal images of U2OS cells treated with either non-targeting (WT) or NOMO-targeting (siNOMO) RNAi. (B) Representative immunofluorescence images of U2OS cells treated with non-targeting siRNA (Ctrl) or NOMO-targeting siRNA (siNOMO), with indicated rescue constructs transfected in lower panels. (C) Immunoblot of cell lysates from experimental setups as in B. (D) Plot of mean ± SD of ER voids area normalized to total ER area determined by calreticulin staining under indicated conditions; n = 131 cells (Ctrl), n = 137 cells (siNOMO), n = 116 cells (siNOMO + F-NOMOr), and n = 115 cells (siNOMO + F-NOMOr^4-Mut). (E and F) FRAP data showing means ± SD of fluorescence intensities of bleached regions normalized to the pre-bleach intensity for each condition. n ≥ 30 cells across a N ≥ 3 biological replicates. Note that NOMO^WT data are the same in E and F. (G) Summary of mean tau (τ) values for each condition corresponding to FRAP experiments shown in E and F. Statistical analyses were performed using an ordinary one-way ANOVA test; ****P < 0.0001 and ns, not significant. Scale bars, 10 μm. Source numerical data and unprocessed blots are available in source data. Source data are available for this figure: SourceData F2.