Figure 6.

Phosphorylated ZFP36L1 interacts with 14-3-3. (A) GST pull-down analysis. E. coli-expressed GST-14-3-3 or GST were bound on glutathione-Sepharose beads. The beads were incubated with 3T3-L1 cell lysates after induction of differentiation for 0, 30 min, 1 h, and 2 h. The associated proteins were separated using SDS-PAGE and immunoblotted with anti-ZFP36L1 and anti-β-tubulin. (B) Activation of ERK and AKT during 3T3-L1 differentiation. Two-day post-confluent 3T3-L1 cells were induced with FMDI for 0, 10, 20, 40, and 60 min. The WCEs were immunoblotted with anti-p-ERK, anti-p-AKT, anti-p-p38, anti-ERK, anti-AKT, anti-p38 and anti-β-tubulin. (C) Interaction between ZFP36L1 and 14-3-3 is blocked by inhibitors of ERK and AKT signaling pathways. Two-day post confluent 3T3-L1 cells were induced with FMDI in the presence of 10 μM U0126, 100 nM wortmannin, or both for 30 min. The cell extracts were isolated for GST pull-down assays, and the pulled-down protein complexes were analyzed by immunoblotting with anti-ZFP36L1. One-tenth input of cell extracts was assessed with anti-ZFP36L1, anti-p-ERK, anti-ERK, anti-p-AKT, and anti-AKT. A representative of three independent experiments with similar results is shown. (D) Inhibition of ERK and AKT signaling pathways downregulates Mkp-1 RNA stability. Two-day post-confluent 3T3-L1 cells were untreated or pretreated with U0126 and wortmannin for 30 min, and then differentiation was induced for 15 min in the presence or absence of U0126 and wortmannin. Subsequently, 10 μg/ml of actinomycin D was added for 0, 10 min, and 20 min to block transcription, and RNA was isolated for quantitative PCR. The expression level of Mkp-1 mRNA under non-treatment of actinomycin D was shown (left panel). The regression curves and Mkp-1 mRNA half-lives were presented (right panel). *P < 0.05, ***P < 0.001.