Figure 7.

ACLP-Ins40 is not secreted from cells and induces ER stress. A, 293 cells were transiently transfected with pCMV6-ACLP or pCMV6-ACLP-Ins40 plasmids, and total protein and conditioned media was collected after 24 h. Western blotting was performed on 25 µg of total protein and equal volumes of 50-fold concentrated conditioned media with antibodies against ACLP. Both ACLP-WT and ACLP-Ins40 proteins were detected in cell lysates, and ACLP but not the ACLP-Ins40 was detected in the conditioned media. B, ACLP and ACLP-Ins40 C-terminal FLAG-tagged vectors were transfected into 3T3 fibroblasts, and ACLP was detected with a Cy3-conjugated anti-FLAG antibody. Cells were counterstained with DAPI (blue). Arrows, perinuclear ACLP expression. C, higher-magnification images of the ACLP-Ins40 protein revealed a pattern consistent with localization to the ER but with apparent absence in the Golgi apparatus indicated by a distinct “patch” adjacent to the nucleus (arrow). D, 3T3 mouse fibroblasts were transiently co-transfected with pCMV6-ACLP-WT-FLAG + XBP1u-EGFP or pCMV6-ACLP-Ins40-FLAG + XBP1u-EGFP plasmids and fixed with 2% paraformaldehyde after 24 h. Cells were stained with a Cy3-conjugated anti-FLAG antibody prior to counterstaining the nuclei with DAPI, mounting, and imaging. Perinuclear expression pattern of ACLP-WT and ACLP-Ins40 proteins in transfected cells is indicated by arrows. E, EGFP expression, indicative of ER stress was quantified from n = 3 independent transfection experiments (p < 0.05). Approximately 10 fields were imaged/condition in each experiment, and a total of 253 ACLP-WT and 320 ACLP-Ins40 cells were scored.