IL-10 and MIP-1α are not involved in the rNef-dependent STAT1 activation in MDMs.

Western blot analysis of phosphotyrosine-STAT1, STAT1, or β-tubulin carried out on total cell extracts from MDMs incubated for indicated times with supernatants from MDMs treated for 2 hours with 100 ng/mL rNef after rNef and IL-10 or MIP-1α double immunodepletions. After rNef treatment, supernatants of MDMs were collected, clarified, and immunodepleted of residual rNef and of IL-10 or MIP-1α. As control, unspecific, species-matched antibodies were added in part of the supernatants. Immunodepleted supernatants were added to fresh MDMs from the same donors and, after incubations of 30 (for MIP-1α only) and 60 minutes, cells were harvested and total cell extracts assayed. Cell extracts from MDMs treated for 2 hours with supernatants from untreated MDMs (zero time) or with 100 ng/mL rNef served as controls. Analyses were performed on cell extracts obtained by pooling simultaneous cell cultures from 4 healthy donors. Specific signals are indicated on the left side; molecular size markers (in kilodaltons) are reported on the right.