Fig. 5.

Nuclear GRP78 regulates EGFR transcription through interaction with ID2. (A) Interaction analysis between GRP78 (HSPA5) and ID2 proteins from the human reference protein interactome mapping project (HuRI). (B) Kaplan–Meier survival analysis of LUAD patients with high and low ID2 expression (n = 719) by KM Plotter. (C) Upper: schematic drawing of ID2-Myc-FLAG. Lower: HEK293AD cells were transfected with the ID2-Myc-FLAG and co-immunoprecipitation assays were performed using IgG or anti-FLAG antibodies. The immunoprecipitated proteins were probed for GRP78 and Myc(ID2). (D) Schematic diagram of the proximity ligation assay (PLA). (E) Representative confocal fluorescence images of PLA between GRP78(WT)-GFP or GRP78(NLS Mut)-GFP and ID2-Myc-FLAG, using antibodies against GFP and FLAG. DAPI (blue) represents nuclei staining, and yellow indicates colocalization. (Scale bars, 10 μm.) (F) Upper: Schematic diagram of ID2-Myc. Lower: HEK293AD cells transfected with ID2-Myc expression construct for 48 h and whole cell lysate was subjected to Western blot for GRP78, EGFR and ID2-Myc proteins with GAPDH serving as loading control. Quantitation of the relative EGFR protein levels normalized to GAPDH was graphed on the right (n = 3). (G) Same as in (F) except GRP78 and EGFR mRNA levels were measured by RT-qPCR and quantitation of their relative levels normalized to β-actin was graphed (n = 3). (H) H1975 cells were transfected with EGFR-Luc reporter gene and si78 in combination with empty vector or ID2-Myc or ID2-Myc+F-78(WT) or ID2-Myc+F-78(NLS Mut) as indicated for 48 h. Whole cell lysate was subjected to Western blot for GRP78, EGFR and ID2-Myc proteins with GAPDH serving as loading control. (I) Same as in (H) except EGFR promoter activity was measured by dual luciferase assay. Data are presented as mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s. denotes not significant (Student’s t test). See also SI Appendix, Fig. S5.