Figure 5

MMP induction and Bcl-2 and Bax expression in uninfected or HIV persistently-infected cell lines. H9 and H9/HTLVIII_B (A) and Jurkat and J1.1 (B) cells were exposed to 10 μM H₂O₂, 0.1 μM STS or complete medium for 24 h and harvested to evaluate mitochondrial membrane potential (ΔΨm) by JC-1 staining by flow cytometry. C) H9 and H9/HTLVIII_B cells were exposed to H₂O₂ and STS. After 24 h, cells were washed and lysed with RIPA buffer. Equal amounts of protein (30 μg/sample) were separated by 10% SDS-PAGE and blotted onto nitrocellulose membranes. The blots were probed with anti-Bcl-2 and anti-Bax antibody, revealed using a peroxidase-conjugated anti-IgG and developed using a chemiluminiscence Western blotting detection reagent. Equal loading was checked by analyzing β-actin expression. Films were analyzed with Scion image analysis software (Scion, Frederick, MD) and the Bcl-2/Bax ratio was depicted. D) H9 and H9/HTLVIII_B cells were exposed to H₂O₂ and STS and after 24 h lysates from cytosolic and mitochondrial fractions were prepared by differential centrifugation. Equal amounts of protein (30 μg/sample) were separated by 10% SDS-PAGE and blotted onto nitrocellulose membranes. Blots were then probed with an anti-Bax polyclonal antibody, incubated with a peroxidase-conjugated anti-rabbit secondary antibody and developed using ECL detection reagent. Equal loading was checked by analyzing β-actin (cytosol fraction) and Complex I (mitochondrial fraction) expression.