β-Adrenergic GSK3 inactivation is PKA dependent. ( a ) The PKA consensus sites around Ser21 and <t>Ser9</t> in GSK3α and <t>GSK3β,</t> respectively, are conserved in various species. ( b ) Immunoblot analysis of total and phosphorylated GSK3α and GSK3β in immortalized brown adipocytes treated with 40 μM H89 for 1 h before stimulation with 0.1 μM ISO for additional 15 min. Some of the cells were stimulated with 100 μM 6-MB-cAMP (6-MB) for 15 min. ( c ) Immunoblot analysis of total and phosphorylated GSK3α and GSK3β as well as phosphorylated HSL (Ser660) in iBAT from AdipoQ -caPKA and wild-type mice housed at room temperature. ( d ) Medium glycerol of immortalized brown adipocytes pre-treated with 10 μM SB216763 for 1 h before stimulation with 0.1 μM for 24 h. ( e ) Immunoblot analysis of phosphorylated and total HSL (Ser660), CREB (Ser133) and phosphorylated PKA substrates in immortalized brown adipocytes pre-treated with 10 μM SB216763 for 1 h before stimulation with 0.1 μM ISO for 1 h. Vinculin (tissues) or TFIIB (cells) serves as loading control. Data presented as mean of means +SEM (n = 4). Statistical significance was determined by two-way ANOVA with repeated measures and Tukey's post hoc test for multiple comparisons. *p