Supplemental data

Figure 1

Figure 2

Figure 3

Table 4

Supplementary Figure 1

Subcellular localization of endogenous Stat1. HepG2 and HEK293 cells were studied under five different conditions as in Figure 1: (a) unstimulated; (b) stimulated acutely with IFN-α or IL-6; (c) stimulated acutely for 20 min and then switched to fresh media for 4.5 h; (d) treated with LMB; and (e) pretreated with LMB, stimulated acutely for 20 min, and then switched to fresh media containing LMB for 4.5 h. Stat1 was visualized after fixation with a Stat-specific Ab (8) and a TRITC-labeled secondary Ab after excitation at 550 nm (Cy3). These images were examined with a ×60 objective lens and are representative of the more than 100 cells.

Supplementary Figure 2

G-St3 and G-St3^Y705F expression levels. Whole cell extracts were prepared 24 h after transient expression of G-St3 and G-St3^Y705F in HEK293 cells and immunoprecipitated with a GFP-specific Ab (no. 814-460; Roche). Precipitates were fractionated by SDS-PAGE and immunoblotted with a Stat3-specific Ab (C20).

Supplementary Figure 3

Time course of dephosphorylation for GFP-Stat3 and 306–318 NES mutants (G-St3 and G-St3^306M) expressed in HEK293 cells. Whole cell extracts were prepared from HEK293 cells transiently (i.e., 24 h) expressing G-St3 and G-St3^306M after the following treatments: (a) no treatment (t = 0); (b) stimulated with IFN-α (1,000 U/ml, t = 20 min); (c) stimulation with IFN-α (1,000 U/ml, 20 min) and then switched to fresh media for 90 min, 150 min, 270 min, and 16 h. Extracts were fractionated by SDS-PAGE and immunoblotted with either a phospho-Stat3 (Ptyr-Stat3) or Stat3 (C20) Ab’s as indicated. The position of exogenous GFP-Stat3 (i.e., G-St3 and G-St3^306M) and endogenous Stat3 are indicated in the right margin. Molecular-weight markers are indicated in the left margin.

Files in this Data Supplement:

• - Figure 1
• - Figure 2
• - Figure 3
• - Table 1