Figure 6. Effects of connector enhancer of kinase suppressor of Ras1 (CNKSR1) overexpression on epidermal growth factor receptor (EGFR)/HER2 signaling in SKBR-3 cells.
(A, B) Western blots of SKBR-3 lysates stably expressing Myc-CNKSR1 (wild-type or ΔPH) or non-tagged CNKSR1 (wild-type or W493A). pEGFR, phosphorylated EGFR (Tyr1068); pHER2, phosphorylated HER2 (Tyr1221/1222). The ratio of band intensities for pEGFR/EGFR or pHER2/HER2, normalized to control, was shown. (C, D) Trypsinized SKBR-3 cells stably expressing Myc-CNKSR1 (wild-type or ΔPH) or non-tagged CNKSR1 (wild-type or W493A) (total 0.5 × 105 cells) were plated. Cell number was counted 72 h after replating. Data are mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01. (E) SKBR-3 cells stably expressing non-tagged CNKSR1 (wild-type or W493A) were transfected with the indicated siRNAs. Cell lysates were prepared at 72 h post-siRNA transfection and subjected to Western blots. pEGFR, phosphorylated EGFR (Tyr1068); pHER2, phosphorylated HER2 (Tyr1221/1222). The ratio of band intensities for pEGFR/EGFR or pHER2/HER2, normalized to control, was shown. (F) SKBR-3 cells were transfected with the indicated siRNAs. At 72 h post-siRNA transfection, serum-starved SKBR-3 cells were treated with EGF (100 ng/ml) for the indicated time. Cell lysates were then prepared and subjected to Western blots. pEGFR, phosphorylated EGFR (Tyr1068); pHER2, phosphorylated HER2 (Tyr1221/1222); pAkt, phosphorylated Akt (Ser473); pERK-1/2, phosphorylated ERK-1/2 (Thr202/Tyr204). The ratio of band intensities for pEGFR/EGFR, pHER2/HER2, pAkt/Akt, or pERK-1/2/ERK1/2, normalized to control, was shown. (G) Scheme of existence of EGFR phosphatases regulated by the RhoB-GTP/CNKSR1 axis. PM, plasma membrane.
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