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. 2004 Feb;24(4):1540–1559. doi: 10.1128/MCB.24.4.1540-1559.2004

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Copyright © 2004, American Society for Microbiology

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FIG. 7. — Expression of EGF receptor and EGFR signaling in telomerase-immortalized fibroblasts expressing v-Fos. (A) Western blotting for expression of EGFR and activated EGFR (pEGFR) in Tif-puro and Tif-Fos cells (100 μg of protein) in the absence and presence of EGF (100 ng/ml for 10 min). A431 cells (25 μg of protein) were used as a positive control, and equal loading was determined using anti-ERK2. (B) Tif-puro and Tif-Fos cells incubated in low (0.2%) serum (LS) for 60 h were stimulated for 15 min with EGF (100 ng/ml), and phosphorylation of ERK was detected with the p44/p42 mitogen-activated protein kinase phosphospecific antibody (pERK1 and pERK2). EGFR-mediated signaling to ERK was inhibited by a 10-min pretreatment with the EGFR-specific inhibitor tyrphostin AG1478 (0.5 and 1.25 μM). Total ERK2 is shown as a loading control. (C) Suppression of EGFR expression by RNA interference. Tif-Fos cells were nucleofected with either scrambled siRNA or EGFR siRNA (100 nM). After 2 days, the cells were lysed and EGFR expression was determined by Western blotting. Parallel cultures of cells were trypsinized and incubated in an in vitro invasion assay for 3 days, and EGFR expression was again determined by Western blotting. Tif-Fos cells were also infected with retroviruses encoding pSUPER or pSUPER. EGFR and EGFR expression was determined by Western blotting in two independent polyclonal populations of each. A431 cells were used as a positive control, and ERK2 expression was determined as a loading control. (D) Tif-puro and Tif-Fos cells were nucleofected with siRNA. After 48 h, proliferation was assessed by counting the attached cells in the absence or presence of 100 nM EGFR siRNA, 0.5 μM AG1478, or 100 ng of EGF per ml. (E) Tif-puro and Tif-Fos cells were stained for actin stress fibers with phalloidin-FITC in the absence or presence of EGF (100 ng/ml) for 24 h with or without pretreatment (10 min) with AG1478 (1.25 μM). Fos expression was detected with the 9E10 anti-Myc monoclonal antibody and anti-mouse-TRITC. Bar, 100 μm. (F) Tif-Fos cells transiently expressing scrambled or EGFR siRNA (100 nM for 72h) or stably expressing pSUPER or pSUPER EGFR were stained with phalloidin-FITC in the absence or presence of EGF (100 ng/ml) for 24 h. Fos expression was detected with the 9E10 anti-Myc monoclonal antibody and anti-mouse-TRITC. Bar, 100 μm. (G) Invasion of Tif-Fos in the absence (black bars) or presence of AG1478 (0.5 and 1.25 μM, gray bars) with or without EGF (100 ng/ml) for 3 days. Invasion was quantitated as described in Materials and Methods and normalized with respect to invasion of Tif-Fos alone. The error bars represent the standard deviation of the mean change. (H) Invasion of Tif-Fos cells transiently expressing scrambled (black bars) or EGFR (gray bars) siRNA 2 days after nucleofection, in the absence or presence of EGF (100 ng/ml) for 3 days or stably expressing pSUPER (black bars) or pSUPER EGFR (gray bars). Invasion is normalized with respect to invasion of Tif-Fos alone, and the error bars represent the standard deviation of the mean change.

FIG. 7. — Expression of EGF receptor and EGFR signaling in telomerase-immortalized fibroblasts expressing v-Fos. (A) Western blotting for expression of EGFR and activated EGFR (pEGFR) in Tif-puro and Tif-Fos cells (100 μg of protein) in the absence and presence of EGF (100 ng/ml for 10 min). A431 cells (25 μg of protein) were used as a positive control, and equal loading was determined using anti-ERK2. (B) Tif-puro and Tif-Fos cells incubated in low (0.2%) serum (LS) for 60 h were stimulated for 15 min with EGF (100 ng/ml), and phosphorylation of ERK was detected with the p44/p42 mitogen-activated protein kinase phosphospecific antibody (pERK1 and pERK2). EGFR-mediated signaling to ERK was inhibited by a 10-min pretreatment with the EGFR-specific inhibitor tyrphostin AG1478 (0.5 and 1.25 μM). Total ERK2 is shown as a loading control. (C) Suppression of EGFR expression by RNA interference. Tif-Fos cells were nucleofected with either scrambled siRNA or EGFR siRNA (100 nM). After 2 days, the cells were lysed and EGFR expression was determined by Western blotting. Parallel cultures of cells were trypsinized and incubated in an in vitro invasion assay for 3 days, and EGFR expression was again determined by Western blotting. Tif-Fos cells were also infected with retroviruses encoding pSUPER or pSUPER. EGFR and EGFR expression was determined by Western blotting in two independent polyclonal populations of each. A431 cells were used as a positive control, and ERK2 expression was determined as a loading control. (D) Tif-puro and Tif-Fos cells were nucleofected with siRNA. After 48 h, proliferation was assessed by counting the attached cells in the absence or presence of 100 nM EGFR siRNA, 0.5 μM AG1478, or 100 ng of EGF per ml. (E) Tif-puro and Tif-Fos cells were stained for actin stress fibers with phalloidin-FITC in the absence or presence of EGF (100 ng/ml) for 24 h with or without pretreatment (10 min) with AG1478 (1.25 μM). Fos expression was detected with the 9E10 anti-Myc monoclonal antibody and anti-mouse-TRITC. Bar, 100 μm. (F) Tif-Fos cells transiently expressing scrambled or EGFR siRNA (100 nM for 72h) or stably expressing pSUPER or pSUPER EGFR were stained with phalloidin-FITC in the absence or presence of EGF (100 ng/ml) for 24 h. Fos expression was detected with the 9E10 anti-Myc monoclonal antibody and anti-mouse-TRITC. Bar, 100 μm. (G) Invasion of Tif-Fos in the absence (black bars) or presence of AG1478 (0.5 and 1.25 μM, gray bars) with or without EGF (100 ng/ml) for 3 days. Invasion was quantitated as described in Materials and Methods and normalized with respect to invasion of Tif-Fos alone. The error bars represent the standard deviation of the mean change. (H) Invasion of Tif-Fos cells transiently expressing scrambled (black bars) or EGFR (gray bars) siRNA 2 days after nucleofection, in the absence or presence of EGF (100 ng/ml) for 3 days or stably expressing pSUPER (black bars) or pSUPER EGFR (gray bars). Invasion is normalized with respect to invasion of Tif-Fos alone, and the error bars represent the standard deviation of the mean change.