Detection of FKHRL1 in primary erythroid progenitor cells and erythroblasts.
(A) CFU-E cells were obtained as described in “Materials and methods.” Total cellular RNA extracted was reverse transcribed and amplified by RT-PCR (see “Materials and methods”). The PCR products were digested with HhaI and resolved by agarose/formaldehyde gel electrophoresis and the gel was stained with ethidium bromide. (B) Fraction 1 (lanes 1 and 5), fraction 2 (lanes 2 and 6), fraction 3 (lanes 3 and 7), and fraction 4 (lanes 4 and 8) were obtained as described in “Materials and methods” and Table 1. After solubilization, cell extracts (2 × 106 cells/1 lane) were resolved by 7.5% or 15% SDS-PAGE and immunoblotted with anti-FKHRL1 antibody (left panel) or anti-FasL antibody (right panel). (C) Phosphorylation of Akt by EPO stimulation in CFU-E cells. CFU-E cells were obtained as described in “Materials and methods” and subsequently cultured without EPO for 2 hours. The cells were then stimulated with EPO (10 U/mL) for the periods indicated. After solubilization, cell extracts (2 × 106 cells/1 lane) were resolved by 7.5% SDS-PAGE and immunoblotted with Akt antibodies directed against phospho-T308 (top panel) or phospho-S473 (middle panel). The blot was reprobed with anti-Akt antibody to confirm equal loading of protein (bottom panel). (D) Phosphorylation of FKHRL1 by EPO stimulation in CFU-E cells. CFU-E cells were prepared as described above. Cell extracts (2 × 106 cells/1 lane) were resolved by 7.5% SDS-PAGE and immunoblotted with FKHRL1 antibodies directed against phospho-T32 (top panel) or phospho-S253 (middle panel). The blot was reprobed with anti-FKHRL1 antibody to confirm equal loading of protein (bottom panel).
