FIGURE 8.

GSK3 associates with STAT3. A, GSK3α or GSK3β were immunoprecipitated (IP) from cell lysates of primary astrocytes treated for 30 min with 1 ng/ml IFNγ, and immunoprecipitated lysates were immunoblotted for STAT3 and GSK3α/β. The level of STAT3 associated with each isoform of GSK3 was evaluated, and values represent the mean ± S.E. (n = 3). STAT3 was immunoprecipitated from membrane (B) or nuclear fractions (C) prepared from primary astrocytes treated for 30 min with 1 ng/ml IFNγ, 100 ng/ml LPS, or both, and immunoprecipitated lysates were immunoblotted for GSK3α/β and STAT3 (n = 4). To ensure the efficiency of the immunoprecipitation, the recovery in the supernatant after immunoprecipitation (Sup.) was performed. D, the IFNγ receptor α-chain (CD119) or STAT3 (E) was immunoprecipitated from membrane fractions prepared from mouse primary astrocytes after infection with adenovirus for the expression of green fluorescent protein (control), constitutively active S9A-GSK3β, or constitutively active S21A-GSK3α, without or with constitutively active FLAG-tagged STAT3C (Ad5STAT3C), for 48 h, and immunoprecipitated lysates were immunoblotted for GSK3α/β, CD119, or STAT3. (n = 3). To ensure the specificity and the efficiency of the immunoprecipitation, an immunoprecipitation with a nonspecific isotypic IgG and the recovery in the supernatant after immunoprecipitation (Sup) were performed. F, astrocytes were treated with control siRNA (Ctl) or siRNA for GSK3α and GSK3β for 48 h followed by a pretreatment for 2 h with 10 ng/ml leptomycin B (LMB) and then addition of IFNγ (1 ng/ml) for 2 h, and nuclear fractions were immunoblotted. The level of phospho-Tyr⁷⁰⁵-STAT3 (PY-STAT3) was calculated, and values represent the mean ± S.E. (n = 5). Immunoblots were reblotted with CREB to ensure equal protein loading.