Journal: The Journal of Biological Chemistry
Article Title: Transforming growth factor β (TGFβ) induces NUAK kinase expression to fine-tune its signaling output
Figure Lengend Snippet: NUAK1 and NUAK2 are transcriptionally induced by TGFβ. A , real-time qRT-PCR analysis of NUAK1 mRNA normalized to HPRT1 mRNA from AG1523 cells after treatment with cycloheximide (20 μ m ) or an equivalent volume of PBS as negative control for 1 h followed by TGFβ (1 ng/ml) stimulation for 5 h. On the right , corresponding immunoblot for NUAK1, phospho-SMAD2 and total SMAD2 under the conditions used in the samples for real-time qRT-PCR. Molecular size markers in kDa are shown. One representative experiment of two is shown. B , real-time qRT-PCR analysis of Nuak2 mRNA normalized to Gapdh mRNA from NMuMG cells after treatment with vehicle or cycloheximide for 15 min followed by TGFβ (5 ng/ml) stimulation for 1 h. Immunoblots of NUAK2, phospho-SMAD2, and total SMAD2 proteins serve as controls for the RNA analysis. SMAD2 serves as a protein-loading control. Molecular size markers in kDa are shown. C , NMuMG cells were pretreated with vehicle or actinomycin D for 1 h before treatment with TGFβ (5 ng/ml) for 1 h. Nuak2 mRNA was normalized to Gapdh mRNA as measured by real-time qRT-PCR. D , immunoblotting of NUAK1 and SMAD4. AG1523 cells were stimulated with TGFβ (1 ng/ml) for 5 h. Ponceau-S staining of the immunoblot serves as a protein-loading control. Molecular size markers in kDa are shown. E , mRNA expression of Nuak2 , Gadd45 γ, and Smad4 in NMuMG cells after treatment with control or Smad4 siRNA and TGFβ (5 ng/ml) stimulation for 1 h. F , immunoblots of NUAK2, PAI-1, SMAD4, and β-actin proteins in MDA-MB-468 cells transiently infected with Adex-LacZ or Adex-SMAD4 (the latter at two different multiplicities of infection, 1 and 4) prior to cell starvation and stimulation with TGFβ (5 ng/ml) for 24 h. β-Actin serves as protein-loading control; a star shows nonspecific protein bands. Molecular size markers in kDa are shown. G , immunoblotting of NUAK1 and NUAK2 in AG1523 cells after treatment with TGFβ (5 ng/ml) for 3 h in the presence of TβRI kinase inhibitors LY2157299 (5 μ m ) or SB505124 (2.5 μ m ) or DMSO (0.1%). Molecular size markers in kDa are shown. H , immunoblots of NUAK2, phospho-ERK1/2, phospho-SMAD2, phospho-SMAD3, SMAD4, and β-actin proteins in NMuMG cells serum-starved overnight, followed by pretreatment with the indicated inhibitors or vehicle (DMSO) for 1 h prior to stimulation with TGFβ (1 ng/ml) for 6 h. β-Actin serves as protein-loading control. Molecular size markers in kDa are shown. Data are presented as mean ± S.E. ( error bars ) after performing at least three independent experiments. Asterisks imply significant differences compared with controls: *, p
Article Snippet: TβRI kinase inhibitor GW6604 was used at a final concentration of 3 μm and was synthesized by the Ludwig Cancer Research Ltd. TβRI kinase inhibitor LY2157299 (Cayman Chemical Co., Stockholm, Sweden) was used at a final concentration of 5 μm , whereas TβRI kinase inhibitor SB505124 (Sigma-Aldrich Sweden AB) was applied at a concentration of 2.5 μm .
Techniques: Quantitative RT-PCR, Negative Control, Western Blot, Staining, Expressing, Multiple Displacement Amplification, Infection