Coexpression of Bmx induces activation of Stat1. (A) COS cells were transfected with Stat1α expression vector together with Bmx, Jak1, Jak2, or control expression vectors, as indicated. Stat1α was immunoprecipitated from cell lysates using anti-HA antibody. The immunoprecipitates were electrophoresed in 7.5% SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-phosphotyrosine antibody. Shown in the lower panel is an immunoblot from total cell lysates electrophoresed in 7.5% SDS-PAGE blotted with anti-Stat1 antibody. (B) COS cells were transfected with Stat1α expression vector together with Bmx, Jak1, Jak2, or control expression vectors, as shown. Eight micrograms of protein from each lysate of transfected cells was incubated with 32P-labeled GAS oligonucleotide and analyzed in the mobility shift assay in 4.5% TBE-PAGE. The Bmx, Jak1, and Jak2 lysates were also supershifted using anti-HA antibody (for Stat1α-HA). Shown in the lower panel is an immunoblot from total cell lysates electrophoresed in 7.5% SDS-PAGE blotted for Stat1α using anti-HA antibody. (C) COS cells were transfected with Stat1α expression vector alone or together with Bmx-HA, Syk, Fyn, or c-Src expression vectors. Lysates were analyzed in the mobility shift assay as above. Shown in the lower panels are immunoblots from total cell lysates electrophoresed in 7.5% SDS-PAGE blotted with anti-phosphotyrosine antibody and anti-Stat1 antibody. The autophosphorylated Syk, Fyn, and c-Src polypeptides are indicated by asterisks.
