Journal: PLoS ONE
Article Title: Phosphorylation of mRNA Decapping Protein Dcp1a by the ERK Signaling Pathway during Early Differentiation of 3T3-L1 Preadipocytes
Figure Lengend Snippet: Dcp1a is phosphorylated during early differentiation of 3T3-L1 preadipocytes. (A) The expression profile of endogenous Dcp1a during the early differentiation of 3T3-L1 preadipocytes. Two days after reaching confluency, cultures of 3T3-L1 preadipocytes were induced to differentiate with an induction cocktail for 0, 1, 2, 4, 8, and 16 h. Cell extracts were isolated, and western blot analysis for detection of Dcp1a protein was conducted using 30 µg of each sample. Tubulin served as the protein input control. (B) CIP treatment. Confluent cultures of 3T3-L1 preadipocytes were induced to differentiate for 0–16 h, and cell extracts were isolated and treated with CIP. Western blot analysis was performed as in panel A. (C) Two days after reaching confluency, 3T3-L1 preadipocytes were induced to differentiate with individual components of the induction cocktail (FBS, MIX, DEX, insulin) or with the induction cocktail (FMDI) or non-induced control (NI). After incubating for 2 h, cell extracts were analyzed as in panel A. (D) Two days after reaching confluency, 3T3-L1 preadipocytes were pretreated with either DMSO (vehicle control) or 20 µM U0126 for 30 min before induction and then were induced with FMDI for 0, 1, 4, and 8 h in DMSO or U0126. Cell extracts were isolated for western blot analysis using anti-Dcp1a, anti-phospho-ERK (p-ERK), and anti-total ERK (t-ERK). All experiments were independently repeated three to five times with similar results, one of which is shown here.
Article Snippet: Two days after reaching confluency, 3T3-L1 cells were stimulated to undergo differentiation into adipocytes by replacing fresh medium with an induction cocktail, FMDI, that was comprised of 10% FBS (HyClone-Characterized), 0.5 mM MIX (Sigma-Aldrich), 5 µM DEX (Sigma-Aldrich), and 1.7 µM bovine insulin.
Techniques: Expressing, Isolation, Western Blot