Figure 2.
Figure 2. Established U-937 sublines constitutively express exogenous Stat1Ser727Ala. (A) The level of Stat1 protein in pCIneo, Stat1.2, Ser727Ala.4, and Ser727Ala.5 cells was determined by Western blot using antibodies directed against total Stat1. The presence of exogenously expressed Stat1Ser727Ala protein was confirmed by Western blot analysis using antibodies specific for the Glu-Glu tag. (B-C) U-937 sublines expressing wt Stat1 (Stat1.2), empty vector (pCIneo), or Stat1Ser727Ala (Ser727Ala.4, Ser727Ala.5) were incubated with ATRA for 30 minutes; lysates were prepared and analyzed by Western blot using antibodies directed against Ser727-phosphorylated Stat1. A lysate of Stat1.2 cells induced by 100 U IFN-γ for 30 minutes was included as a positive control for Stat1 Ser727 phosphorylation (*). Quantitation of Stat1 Ser727 phosphorylation, normalized to actin expression, is shown in the lower panels (fold induction).

Established U-937 sublines constitutively express exogenous Stat1Ser727Ala. (A) The level of Stat1 protein in pCIneo, Stat1.2, Ser727Ala.4, and Ser727Ala.5 cells was determined by Western blot using antibodies directed against total Stat1. The presence of exogenously expressed Stat1Ser727Ala protein was confirmed by Western blot analysis using antibodies specific for the Glu-Glu tag. (B-C) U-937 sublines expressing wt Stat1 (Stat1.2), empty vector (pCIneo), or Stat1Ser727Ala (Ser727Ala.4, Ser727Ala.5) were incubated with ATRA for 30 minutes; lysates were prepared and analyzed by Western blot using antibodies directed against Ser727-phosphorylated Stat1. A lysate of Stat1.2 cells induced by 100 U IFN-γ for 30 minutes was included as a positive control for Stat1 Ser727 phosphorylation (*). Quantitation of Stat1 Ser727 phosphorylation, normalized to actin expression, is shown in the lower panels (fold induction).

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