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. Author manuscript; available in PMC: 2012 Jul 1.
Published in final edited form as: Biochim Biophys Acta. 2011 Jun 13;1809(7):306–315. doi: 10.1016/j.bbagrm.2011.05.016

Fig. 4. Expression of mRNA and protein in SW480 after U0126 treatment.

Fig. 4

a) mRNA expression after U0126 treatment. SW480 cells were treated with either control DMSO (MEM), MEM-AA, or MEM-AA containing 1.25, 2.5, or 5 μmol/L of the MEK1/2 inhibitor U0126 for 8 h. Relative mRNA level was determined by quantitative real time RT-PCR and normalized to DMSO control. b) Western blot analysis after U0126 treatment. ATF4, p-eIF2α, and p-ERK proteins were analyzed by Western blot analysis in SW480 after U0126 treatment. Total ERK and total eIF2α were also analyzed to ensure that the changes observed for p-ERK and p- eIF2α are specific for phosphorylated forms. c) Protein abundance after U0126 treatment. Protein level of ATF4, p-eIF2α, and p-ERK was quantified after Western blot analysis in SW480 cells treated with U0126. A non-specific band was used as the loading control to normalize the raw data. Each data point represents the mean ± S. D. of at least three independent samples. For data from the U0126 treated samples, asterisks (*) represent statistical significance compared to values in amino acid deprived condition (MEM-AA, P < 0.05). d) DKK1 protein expression. The MEM-, MEM-, and MEM-AA+U0126-treated cells were fixed on glass cover slips and subsequently used for immunofluorescent staining. DKK1 protein was analyzed after immunofluorescent staining using an antibody against DKK1 protein and an Alexa Fluor 647-labeled secondary antibody (red). The cover slips were also counterstained with Hoechst 33342 fluorescent staining for nucleus (blue). The two pictures were overlaid to show DKK1 staining on top of the nuclear staining (merged).