Journal: The Journal of biological chemistry

Article Title: Modulation of epidermal growth factor receptor gene transcription by a polymorphic dinucleotide repeat in intron 1.

doi: 10.1074/jbc.274.19.13176

Figure Lengend Snippet: FIG. 3. In vitro run-off transcription of the EGFR gene. A, EGFR 4,050-bp PCR products from the indicated cell lines were used as templates for run-off transcription with A431 nuclear extracts. After transcription, DNA was digested with PstI, electrophoresed through a denaturing 4% polyacrylamide, 13 TBE gel, and transferred to a nylon membrane. RNA products and DNA fragments were detected after hybridization with a fluoresceinated RNA probe. The DNA template is detected in fragments of 1,051, 848, 706, and 412 nt, and run-off transcripts are sized approximately 1,650 and 1,550 nt. The sizes of an RNA standard are shown on the left side. Controls without DNA tem- plate and with RNase treatment in the right lanes revealed no RNA products. B, determination of the ratio of RNA product intensity to the DNA 1,051-bp band intensity dependent on the DNA template amount. The ratio is approximately constant between 10 and 100 ng DNA.

Article Snippet: Approximately 50 ng of PCR product was combined with 6 ml of 13 transcription buffer (20 mM HEPES (pH 7.9), 100 mM KCl, 0.5 mM dithiothreitol, 0.2 mM EDTA, 20% glycerol, in diethyl pyrocarbonate-treated water), 1 ml of RNasin, 2 ml of 50 mM MgCl2, 1 ml of 10 mM NTP mix (all Promega), 5 ml of A431 nuclear extract (25 mg of protein, Santa Cruz Biotechnology), and diethyl pyrocarbonate-treated water to a final volume of 25 ml.

Techniques: In Vitro, Membrane, Hybridization

Journal:

Article Title: Enteropathogenic Escherichia coli -Induced Epidermal Growth Factor Receptor Activation Contributes to Physiological Alterations in Intestinal Epithelial Cells ▿

doi: 10.1128/IAI.01690-06

Figure Lengend Snippet: EPEC-induced EGFR activation is delayed in polarized epithelial cells. (A) Polarized C2BBE cells grown on permeable supports were infected with WT EPEC, and the extracts isolated at specific time points were immunoblotted against EGFR pY1068. EGF-treated A431 cell extract was included as a positive control to confirm the identity of the band. (B) Data from three similar experiments were evaluated by densitometry (P < 0.005 by analysis of variance for 240 min).

Article Snippet: Control EGF-treated A431 cell extracts and antibody to total EGFR were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Infection, Isolation, Positive Control