Fig. 4.

8ab up-regulates the synthesis of ER-resident chaperons. (A) 8ab protein activates GRP78 promoter. HeLa cells were transiently co-transfected with pCI8abHA and a reporter plasmid containing firefly luciferase gene under the control of GRP78 promoter for 40 h (gray bar). Control cells were co-transfected with reporter plasmids and empty pCI vector for 24 h, and then either treated or untreated with TG 500 nM for an additional 16 h (black or white bar). In each case, pRL-TK encoding Renilla luciferase was co-transfected as an internal control. The cell lysates were harvested and assayed for firefly and Renilla luciferase activities. The results were normalized with Renilla luciferase activities and were averaged with S.E. of three experiments. (B). Indirect immunofluorescence analysis of GRP78 in 8ab-expressing cells. Vero E6 cells were transiently transfected with pCI8abHA for 30 h, and labeled with anti-HA and anti-GRP78 antibodies. Bound primary antibodies were revealed with Alexa Fluor 568- and Alexa Fluor 488-conjugated secondary antibodies, respectively. (C) Immunoblotting analysis of ER-resident chaperons in transfected cells. HeLa cells were transiently transfected with pCI-8abHA for 30 h, or pCI for 24 h, then treated or untreated with TG 500 nM for an additional 6 h. Cell lysates were used for Western blot analysis using anti-KDEL (for GRP78 and GRP94) and anti-calreticulin antibodies. Expression level of actin was used as loading control.