(A) Levels of pRb during neutrophilic and monocytic differentiation on days 0 and 3. Cells selected for CD34 expression were incubated on day 0 with cytokines favoring neutrophilic (G-CSF and SCF, both at 50 ng/mL) or monocytic (FL at 50 ng/mL and IL-3 at 25 ng/mL) differentiation, respectively. On day 0, 0.55 × 106 cells not stimulated with any cytokines were subjected to protein extraction, followed by specific immunoprecipitation with an anti-pRb antibody (sc-102; Santa Cruz), 6% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and Western blot analysis. On day 3, 0.55 × 106 cells from cells incubated with G-CSF/SCF or FL/IL-3, respectively, were subjected to protein extraction, followed by specific immunoprecipitation with an anti-pRb antibody (sc-102; Santa Cruz), 6% SDS-PAGE, and Western blot analysis. pRb of different molecular weights are indicated with arrows (ppRb, hyperphosphorylated; pRb, hypophosphorylated). (B) Levels of pRb during neutrophilic and monocytic differentiation on days 3 and 5. Cells selected for CD34 expression were incubated on day 0 with cytokines, favoring neutrophilic (G-CSF and SCF, both at 50 ng/mL) or monocytic (FL at 50 ng/mL and IL-3 at 25 ng/mL) differentiation, respectively. After 3 and 5 days of expansion, respectively, 1.5 × 106 cells were subjected to protein extraction, followed by specific immunoprecipitation with an anti-pRb antibody (sc-102; Santa Cruz), 6% SDS-PAGE, and Western blot analysis. pRb of different molecular weights are indicated with arrows (ppRb, hyperphosphorylated; pRb, hypophosphorylated).
