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. Author manuscript; available in PMC: 2017 Feb 27.
Published in final edited form as: J Biol Chem. 2004 Oct 1;279(49):51075–51081. doi: 10.1074/jbc.M408607200

Fig. 6. GSK-3β inhibits LPA-induced JNK phosphorylation.

Fig. 6

A, the effect of stable re-expression of GSK-3β. GSK-3β −/− cells were co-transfected with pcDNA3-HA-GSK-3β and pcDNA3.1/GS (containing the Zeocin resistance gene). Stable clones (G28 and G60) that expressed HA-GSK-3β were identified by immunoblotting with an antibody against GSK-3α/β and GSK-3β, with wild type MEFs included as a positive control (top). G28, G60, and empty vector control clone C9 were starved and treated with LPA (1 μM) for the indicated periods of time (min) (bottom). Cell lysates were prepared and analyzed by immunoblotting for JNK phosphorylation with a JNK phospho-specific (p-JNK) antibody (bottom). B, effect of the inducible expression of GSK-3β. The EC1214A-GSK3β inducible system involves a single vector carrying both the tetracycline-responsive transcriptional activator (tTA) and a tetracycline responsive element (TRE). The plasmid produces the tTA under the control of the cytomegalovirus promoter, and then tTA binds to TRE, leading to transcriptional activation of GSK3β in the absence of tetracycline. TK, thymidine kinase; BGH, bovine growth hormone; pA, poly(A). A stable clone with minimum expression of GSK-3β in the presence of tetracycline was identified and incubated in the presence (Tet 2 μg/ml) and absence of tetracycline (Tet 0 μg/ml) for 36 h. The cells were stimulated without or with LPA (1 μM) for 30 min and analyzed by immunoblotting for JNK phosphorylation with a JNK phospho-specific (p-JNK) antibody. Induction of GSK-3β expression in the inducible clone following removal of tetracycline was confirmed by immunoblotting with antibody against GSK-3α/β (bottom).