Figure 5.

Stat3β monoclonal antibodies specifically immunoprecipitate Stat3β from protein lysates. Stat3β Mab 516G10H9 (1:30) and Stat3α Ab (D1A5) were added to 200 µg of protein from either untransfected Stat3^−/−murine embryonic fibroblasts (MEFs) or cells transiently transfected with plasmids encoding fusion proteins Stat3α-GFP and Stat3β-GFP, incubated overnight at 4 °C. The lysates were then incubated with protein G-agarose beads for 30 min at 4 °C and after centrifugation, the beads boiled in loading dye and the eluted proteins subjected to SDS-PAGE, transferred to nitrocellulose membrane and probed for total Stat3 (clone 124H6, upper panel). An equivalent volume of cell lysates (20 µL) used in each reaction were subjected to SDS-PAGE, transferred to nitrocellulose membrane and probed for total Stat3 (clone 124H6, lower panel) to indicate the amount of protein input.