Fig 2: AKT1 induced HSF1 activity stronger than other known activating kinases despite similar induction of S326 phosphorylation. (A) Recombinant proteins were incubated at 30 °C for 2 h in the presence of ATP and subsequently subjected to SDS/PAGE and immunoblotting with indicated antibodies. (B‐C) HEK‐293 cells were transfected with vector, AKT1, mTOR, MEK1, p38 or DYRK2 for 48 h. Cells were collected and subjected to immunoblotting with indicated antibodies (B) or luciferase reporter assay (C). S326‐phosphorylated bands in B were subjected to densitometry with relative differences normalized to GAPDH presented for each kinase transfected. (D) HEK‐293 cells were transfected with non‐targeting control siRNA or siRNA directed to AKT1, mTOR, MEK1, p38δ or DYRK2 along with an HSE luciferase reporter. Cells were then incubated at 42 °C for 1 h and subsequently collected and subjected to the luciferase reporter assay. (E‐I) HEK‐293 cells were transfected with control siRNA or siRNA directed at the indicated genes. Total RNA was collected and subjected to RT‐qPCR with the indicated primers. Experiments were completed in triplicate and analysed using one‐way ANOVA and Tukey’s post‐hoc test. Data are presented as mean ± SEM. *P < 0.05.