Fig. 2. LUMA is an ER protein enriched at the INM. (A) Localization of LUMA in relation to emerin or lamin A/C in HeLa and NIH 3T3 cells, visualized by immunofluorescence staining. LUMA colocalizes with emerin at the NE and frames lamin A/C. A non-nuclear ER pool of LUMA is indicated by arrows. Overlay images show LUMA in green, emerin or lamin A/C in red. Scale bars, 5 µm. (B) Wild-type (MEF LMNA^+/+) and LMNA knockout (MEF LMNA^–/–) MEFs were stained with anti-LUMA antibody. Scale bars, 5 µm. (C) Immunoblots of proteins extracted from MEF LMNA^–/– and LMNA^+/+ cells were stained for LUMA, lamin A/C and, as a loading control, tubulin. (D) Untreated HeLa cells or HeLa cells transiently expressing LUMA 1-400 were extracted with Triton X-100 prior to fixation. Emerin and either endogenous LUMA (HeLa) or LUMA 1-400 carrying a V5-tag (HeLa/LUMA 1-400) were visualized by immunofluorescence staining. (E) Scheme of subcellular fractionation employed for detecting LUMA enrichment at the NE. (F) Whole-cell homogenate (H), nuclei (N), nuclear envelopes (NE) and ER membranes (ER) were prepared from N2a cells and immunoblotted for LUMA, emerin, LAP2 membrane isoforms, lamin A/C, calnexin and Sec61 (G) Whole cells, isolated nuclei and nuclear envelopes were extracted with Triton X-100 under low-salt (0.1 M NaCl) or high-salt (2 M NaCl) conditions. Solubilized (S) and pelleted (P) proteins were immunoblotted for LUMA.