Figure 1
Figure 1. Hsp72 augments EBNA2 and EBNALP coactivation. (A) Schematic depiction of Hsp72 and Hsp72 mutants. (B) Hsp72 overexpression augments EBNALP coactivation with EBNA2 of the LMP1 promoter (LMP1p). BJAB B lymphoblasts were transfected with 5 μg LMP1p-Luciferase reporter plasmid, 10 μg EBNA2 expression plasmid (E2 +), 10 μg EBNALP expression plasmid (LP +), 10, 30, or 50 μg Flag-tagged Hsp72 expression plasmid (FHsp72), 2 μg β-gal transfection control expression plasmid, and an appropriate amount of control pSG5 expression vector DNA so that cells were transfected with equal amounts of DNA. Luciferase and β-gal activity activities were measured 24 hours after transfection. Luciferase activities were corrected for β-gal activity. EBNA2, EBNALP, and FHsp72 expression were determined by Western blotting with PE2, JF186, and M2 (Flag epitope specific) antibodies. (C) To assess the effect of Hsp72 overexpression on EBNA2 and EBNALP coactivation with varying levels of EBNALP, BJAB cells were transfected with 5 μg LMP1p-Luciferase reporter plasmid, 10 μg EBNA2 expression plasmid (E2 +), 1, 3, or 5 μg EBNALP (LP +) expression plasmid, 30 μg FHsp72 expression plasmid, or vector DNA to balance plasmid input for each transfection and processed as in panel B. (D) EBV latency I infected Akata BL cells were transfected with 10 μg EBNA2, 10 μg EBNALP, 10, 30, or 50 μg FHsp72, and appropriate amounts of control expression vector. LMP1 expression was assayed by Western blot with S12 mouse monoclonal antibody at 48 hours after transfection. (E) The effect of Hsp40 overexpression on EBNA2 and EBNALP coactivation of the LMP1p was assessed using a protocol identical to that in panel B except for FHsp40 expression vector instead of Fhsp72. (F) The effect of Hsp72 or Hsp72 mutants on EBNA2 and EBNALP coactivation of the LMP1p was assessed using a protocol similar to that used in panel B with 30 μg FHsp72 or FHsp72 mutant expression plasmids. (G) The association of EBNALP with Hsp72 and Hsp72 mutants was assessed in BJAB cells after transfection with 10 μg EBNALP and 10 μg FHsp72 or FHsp72 mutant expression plasmids. Lysates were immune precipitated with M2 Flag epitope–specific beads and Western blotted with JF186 monoclonal antibody to EBNALP. EBNALP input (5%) is shown. Lane 1 (C) is a control IP with M2 antibody in the absence of Flag tagged Hsp72 expression. Error bars indicate one standard deviation from the mean for data from multiple experiments in this and in subsequent figures.

Hsp72 augments EBNA2 and EBNALP coactivation. (A) Schematic depiction of Hsp72 and Hsp72 mutants. (B) Hsp72 overexpression augments EBNALP coactivation with EBNA2 of the LMP1 promoter (LMP1p). BJAB B lymphoblasts were transfected with 5 μg LMP1p-Luciferase reporter plasmid, 10 μg EBNA2 expression plasmid (E2 +), 10 μg EBNALP expression plasmid (LP +), 10, 30, or 50 μg Flag-tagged Hsp72 expression plasmid (FHsp72), 2 μg β-gal transfection control expression plasmid, and an appropriate amount of control pSG5 expression vector DNA so that cells were transfected with equal amounts of DNA. Luciferase and β-gal activity activities were measured 24 hours after transfection. Luciferase activities were corrected for β-gal activity. EBNA2, EBNALP, and FHsp72 expression were determined by Western blotting with PE2, JF186, and M2 (Flag epitope specific) antibodies. (C) To assess the effect of Hsp72 overexpression on EBNA2 and EBNALP coactivation with varying levels of EBNALP, BJAB cells were transfected with 5 μg LMP1p-Luciferase reporter plasmid, 10 μg EBNA2 expression plasmid (E2 +), 1, 3, or 5 μg EBNALP (LP +) expression plasmid, 30 μg FHsp72 expression plasmid, or vector DNA to balance plasmid input for each transfection and processed as in panel B. (D) EBV latency I infected Akata BL cells were transfected with 10 μg EBNA2, 10 μg EBNALP, 10, 30, or 50 μg FHsp72, and appropriate amounts of control expression vector. LMP1 expression was assayed by Western blot with S12 mouse monoclonal antibody at 48 hours after transfection. (E) The effect of Hsp40 overexpression on EBNA2 and EBNALP coactivation of the LMP1p was assessed using a protocol identical to that in panel B except for FHsp40 expression vector instead of Fhsp72. (F) The effect of Hsp72 or Hsp72 mutants on EBNA2 and EBNALP coactivation of the LMP1p was assessed using a protocol similar to that used in panel B with 30 μg FHsp72 or FHsp72 mutant expression plasmids. (G) The association of EBNALP with Hsp72 and Hsp72 mutants was assessed in BJAB cells after transfection with 10 μg EBNALP and 10 μg FHsp72 or FHsp72 mutant expression plasmids. Lysates were immune precipitated with M2 Flag epitope–specific beads and Western blotted with JF186 monoclonal antibody to EBNALP. EBNALP input (5%) is shown. Lane 1 (C) is a control IP with M2 antibody in the absence of Flag tagged Hsp72 expression. Error bars indicate one standard deviation from the mean for data from multiple experiments in this and in subsequent figures.

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