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. 2015 Feb 17;16(2):4343–4361. doi: 10.3390/ijms16024343

Figure 6.

Figure 6

Western blot analysis of STAT1 expression and phosphorylation. Cells were stimulated with IFN-γ for 0, 0.5 or 4 h. Lysates were cleared of cellular debris, and equal concentrations of protein were separated via SDS-PAGE. Proteins were identified by incubating nitrocellulose with antibodies against STAT1 (RGP, VGP, MET; top) or pSTAT1 (RGP, VGP, MET; middle). β-Actin was used as a loading control (RGP, VGP, MET; bottom). (A,B) STAT1 is constitutively expressed and phosphorylated at tyrosine 701 (Y701) following 30 minutes of IFN-γ stimulation in RGP cells; (C,D) STAT1 expression and phosphorylation in VGP cells is similar to RGP; (E,F) STAT1 expression is absent in MET cells. The data shown are representative of at least three experimental replicates. y-axes (B,D,F) represent Total Pixels (Quantification of A,C,E). NS: Not Significant; (G) MET cells + STAT1. MET cells were transfected with 0, 5, 10 µg of Flag-STAT1 for 24 h followed by 48 h of IFN-γ stimulation. Cell surface expression of MHC II was analyzed via flow cytometry; and (H) Expression of Flag-STAT1 following 24 h of incubation with transfection reagent:plasmid complexes. Both 5 µg (light green) and 10 µg (orange) of STAT1 led to restoration of MHC II on the cell surface, compared to non-transfected cells (light blue) and the unstained control (shaded). ** p < 0.005, *** p < 0.001, **** p < 0.0001.