Subcellular Localization of BmGRP78 Under Stress

We investigated the subcellular location of BmGRP78 under different stress-induced conditions using immunofluorescence staining [51 (link),52 (link)] and green fluorescence protein (EGFP)-tagged cloning [53 (link)], as in previous studies, with some modifications, respectively. Briefly, BmN cells were cultured in laser confocal dishes (Solarbio, Beijing, China) at 70–80% confluency (2 × 10⁶ cells/well) and incubated with purified anti-BmGRP78 IgG for 2 h at room temperature in the dark. After rinsing three times in PBST, the cells were incubated with Cy3-labeled goat antirabbit IgG (SA00009-2, Proteintech, Wuhan, China). For EGFP-tagged clone, the BmGRP78 open reading frame was amplified and cloned into the pIEx-1-EGFP vector. The clone containing the appropriate insert segment was identified by sequencing. The positive construct (2 µg) of the BmGRP78 gene was transfected into the BmN cells using Fugene 6 transfection reagent (Promega, Madison, WI, USA). After 24 h of incubation, the BmN cells were fixed with 4% paraformaldehyde for 15 min. Subsequently, the nuclei of the BmN cells were stained with DAPI for 10 min and observed under a confocal microscope (FV1200, Olympus, Tokyo, Japan).