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. 2001 Apr;21(8):2933–2943. doi: 10.1128/MCB.21.8.2933-2943.2001

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Copyright © 2001, American Society for Microbiology

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FIG. 5 — Severe impairment in Srf^−/− ES cells of IEG induction by serum or TPA. (A and B) Northern blot analysis of Egr-1 (A) and c-fos. (B) RNA expression in the Srf^−/+ ES line 44 (top panels) or the Srf^−/− ES line 226-100 (bottom panels) under normal growth conditions (lane 1), after serum withdrawal for 24 h (lane 2), or after serum withdrawal and subsequent induction with either 15% serum (lanes 3 to 6) or 100 ng of TPA/ml (lanes 7 to 10) for the indicated times. Lane 11 represents a positive hybridization control included in the Srf^−/− blot. The Egr-1 blot (A) was reprobed for c-fos (B). In addition, a Fox probe was used to verify equivalent RNA loading in each lane (33). The asterisk (B) indicates a residual Egr-1 signal still present from prior probing of the blot. (C to E) Quantitative RT-PCR measuring expression of Egr-1 (C), c-fos (D), and Vinculin (E) in ES cell lines of the indicated Srf genotype, as well as in Srf^−/− ES cells that ectopically express human Srf cDNA (Srf^{−/−rescue}). mRNA induction factors relative to prestimulation levels are given. Cells were either serum starved for 36 h or serum starved and subsequently restimulated with 15% serum for the indicated times. mRNA levels were normalized to mRNA levels of the Hprt gene, and values are given as fold induction over nonstimulated cells. Each kinetic measurement was performed in duplicate.

FIG. 5 — Severe impairment in Srf^−/− ES cells of IEG induction by serum or TPA. (A and B) Northern blot analysis of Egr-1 (A) and c-fos. (B) RNA expression in the Srf^−/+ ES line 44 (top panels) or the Srf^−/− ES line 226-100 (bottom panels) under normal growth conditions (lane 1), after serum withdrawal for 24 h (lane 2), or after serum withdrawal and subsequent induction with either 15% serum (lanes 3 to 6) or 100 ng of TPA/ml (lanes 7 to 10) for the indicated times. Lane 11 represents a positive hybridization control included in the Srf^−/− blot. The Egr-1 blot (A) was reprobed for c-fos (B). In addition, a Fox probe was used to verify equivalent RNA loading in each lane (33). The asterisk (B) indicates a residual Egr-1 signal still present from prior probing of the blot. (C to E) Quantitative RT-PCR measuring expression of Egr-1 (C), c-fos (D), and Vinculin (E) in ES cell lines of the indicated Srf genotype, as well as in Srf^−/− ES cells that ectopically express human Srf cDNA (Srf^{−/−rescue}). mRNA induction factors relative to prestimulation levels are given. Cells were either serum starved for 36 h or serum starved and subsequently restimulated with 15% serum for the indicated times. mRNA levels were normalized to mRNA levels of the Hprt gene, and values are given as fold induction over nonstimulated cells. Each kinetic measurement was performed in duplicate.