Fig. 6.

Nogo-B receptor (NgBR) knockdown attenuates epidermal growth factor receptor (EGF)-stimulated signaling and estrogen receptor alpha (ERα) phosphorylation in MCF-7-TamR cells. a MCF-7-TamR cells were transfected with siNgBR and treated with EGF (100 ng/mL) for 5 min. Downstream signaling of the EGF pathway was determined using western blot assay. b Quantitative analysis of phosphorylated proteins presented in Fig. 4b were carried out using ImageJ and were normalized to total proteins. c MCF-7-TamR cells were transfected with siNgBR and treated with 100 ng/mL EGF for 12 h. Survivin protein levels were determined using western blot analysis. d Quantitative analysis of survivin protein levels presented in Fig. 4c were carried out using ImageJ and were normalized to β-actin. e NgBR is required for the EGF-stimulated activation of H-Ras and K-Ras in MCF-7-TamR cells. MCF-7-TamR cells were transfected with siNgBR and stimulated with 100 ng/mL EGF for 5 min. The complex of activated Ras (GTP-loaded Ras) was precipitated from total cell lysates using GST-RBD beads. Protein levels were detected by western blotting. Both Ras and NgBR were detected in the complexes precipitated by the Raf-pull-down method. The data are from three separate repeat experiments and are presented as the mean ± SD (*p < 0.05, n = 3)