Stimulation of G1-to-S progression by Na+/H+ exchanger regulatory factor 1 (NHERF1) knockdown. (a) MCF7/NHERF-910 and MCF7/Babe cells were cultured in serum-free media for 1 day to accumulate cells at G1/G0 phase. Cells were then re-fed with medium supplemented with 10% foetal bovine serum for 0, 14, 19, 24, and 29 hours, when cells were trypsinised for fluorescence-activated cell sorting analyses of cell cycle progression. (b) Cells harvested at various time points were lysed for immunoblotting analyses of Rb, p27, cdk2, cdk4, cyclin D1, and cyclin E expression. Hyper-phosphorylated and hypo-phosphorylated forms of Rb are indicated by arrow and arrowhead, respectively. Membranes were also probed with NHERF1 for knockdown verification and with β-actin for loading control. The result presented was representative of two independent experiments.