Journal: Journal of Biological Chemistry

Article Title: The Transforming Growth Factor-β-Smad3/4 Signaling Pathway Acts as a Positive Regulator for TLR2 Induction by Bacteria via a Dual Mechanism Involving Functional Cooperation with NF-κB and MAPK Phosphatase 1-dependent Negative Cross-talk with p38 MAPK

doi: 10.1074/jbc.m602124200

Figure Lengend Snippet: FIGURE5.TGF-signalingalso mediates NTHi-induced TLR2 transcription via MKP-1-dependent neg- ative cross-talk with p38 MAPK. A, pretreatment with SB203580 (1 M) and overexpression of dominant negative mutants of p38 and p38 enhanced NTHi-induced TLR2 expression at mRNA level in HeLa cells. B, pretreatment with SB431542 (1 M) enhanced NTHi-induced phosphorylation of p38, but not MKK3/6, in HeLa cells, as assessed by Western blotting using anti-phosphorylated p38 and MKK3/6 antibody. C, overexpression of a dominant negative mutant of TRI enhanced NTHi-induced phosphorylation of p38, but not MKK3/6, in HeLa cells. D, overexpression of a dominant negative mutant of TRII enhanced NTHi-induced phosphorylation of p38, but not MKK3/6, in HeLa cells. E, TRI and TRII knockdown using TRI-siRNA and TRII-siRNA enhanced NTHi-induced phosphorylation of p38, but not MKK3/6, in HeLa cells. F, overexpression of dominant negative mutants of Smad3 and Smad4 enhanced NTHi-induced phosphorylation of p38, but not MKK3/6, in HeLa cells. G, the addition of exogenous TGF-1 (1 ng/ml) attenuated NTHi-induced phosphorylation of p38, but not MKK3/6, in HeLa cells. H, overexpressing a transdominant mutant form of IB or a dominant negative mutant form of IKK did not alter NTHi- induced p38 phosphorylation. Values are the mean S.D. (n 3).

Article Snippet: Western Blot Analysis—Antibodies against phospho-Smad3, phospho-p38, p38, phospho-MKK3/6, and MKK3 were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Over Expression, Dominant Negative Mutation, Expressing, Phospho-proteomics, Western Blot, Knockdown, Mutagenesis

Journal: BMC Cancer

Article Title: MicroRNA-21 promotes hepatocellular carcinoma HepG2 cell proliferation through repression of mitogen-activated protein kinase-kinase 3

doi: 10.1186/1471-2407-13-469

Figure Lengend Snippet: The expression of MAP2K3 in human HCC tissues determined by IHC

Article Snippet: The membranes were probed with rabbit anti-MAP2K3 antibody and anti-GAPDH antibody (Boster, Wuhan, China) or (1:200, Boster, Wuhan, China) were for the interested protein MAP2K3 and endogenous GAPDH for loading control, respectively.

Techniques: Expressing, Immunohistochemistry

Journal: BMC Cancer

Article Title: MicroRNA-21 promotes hepatocellular carcinoma HepG2 cell proliferation through repression of mitogen-activated protein kinase-kinase 3

doi: 10.1186/1471-2407-13-469

Figure Lengend Snippet: Validation of MAP2K3 mRNA as a target of miR-21. (A) : Sequence of potential binding site of miR-21 in the 3’UTR of MAP2K3 mRNA (top panel), mutations were introduced into the binding site for generation of mutated MAP2K3 3’TUR (bottom panel). ( B and C ): Validation of miR-21 target using MAP2K3 3’UTR luciferase reporter. Cells co-transfected with pMIR-Report/MAP2K3 3’UTR (WT) or pMIR-Report/Mut-MAP2K3 3’UTR (Mut) and pAd/pri-miR-21 (B) , pAd/miR-21/inhibitor (C) , and pAd/con plasmids showed a decreased luciferase activity in pAd/pri-miR-21 cells (B) . Luciferase activity after site directed mutagenesis of the 3’UTR of MAP2K3 mRNA in the miR-21 seed sequence (pMIR-Report/Mut-MAP2K3) was significantly higher with respect to the pMIR-Report/MAP2K3 vector transfected cells ( B and C ). Results represented the mean ± SD from three independent triplicated experiments (N=9).

Article Snippet: The membranes were probed with rabbit anti-MAP2K3 antibody and anti-GAPDH antibody (Boster, Wuhan, China) or (1:200, Boster, Wuhan, China) were for the interested protein MAP2K3 and endogenous GAPDH for loading control, respectively.

Techniques: Biomarker Discovery, Sequencing, Binding Assay, Luciferase, Transfection, Activity Assay, Mutagenesis, Plasmid Preparation

Journal: BMC Cancer

Article Title: MicroRNA-21 promotes hepatocellular carcinoma HepG2 cell proliferation through repression of mitogen-activated protein kinase-kinase 3

doi: 10.1186/1471-2407-13-469

Figure Lengend Snippet: miR-21 targets MAP2K3 mRNA. The HepG2 cells were infected with Ad/pri-miR-21, Ad/miR-21/inhibitor or Ad/con adenoviral vector. The expression of MAP2K3 was detected by immunoblotting analysis against anti-MAP2K3 antibody. Compared with Ad/con group, *: p <0.05. Data in A represented the mean ± SD from three independent triplicated experiments (N=9).

Article Snippet: The membranes were probed with rabbit anti-MAP2K3 antibody and anti-GAPDH antibody (Boster, Wuhan, China) or (1:200, Boster, Wuhan, China) were for the interested protein MAP2K3 and endogenous GAPDH for loading control, respectively.

Techniques: Infection, Plasmid Preparation, Expressing, Western Blot

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Silencing Ras-Related C3 Botulinum Toxin Substrate 1 Inhibits Growth and Migration of Hypopharyngeal Squamous Cell Carcinoma via the P38 Mitogen-Activated Protein Kinase Signaling Pathway

doi: 10.12659/MSM.907468

Figure Lengend Snippet: Silencing Rac1 inhibits the activation of P38 MAPK signal in vitro . The levels of MKK3 and p-MKK3 ( A, B ) and P38 and p-P38 ( C, D ) were detected by Western blot analysis. GAPDH served as the internal reference. All experiments were repeated 3 times and the results are presented as mean ±SD. *** P<0.001 compared with the negative control group.

Article Snippet: The membranes were blocked with 5% skim milk or 1% bovine serum albumin, and then incubated with Rac1 antibody, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1: 1000; Proteintech, Wuhan, China), Cyclin D1 antibody (1: 1000; BOSTER, Wuhan, China), Cyclin E antibody, Cyclin B antibody (1: 500; Bioss, Beijing, China), metal matrix proteinase (MMP)-2 antibody, MMP-9 antibody, B cell lymphoma-2 (Bcl-2) antibody, Bcl-2-associated X protein (Bax) antibody, phosphorylated-MAP kinase (p-MKK3) antibody (1: 500; Sangon Biotech, Shanghai, China), caspase-3 antibody, caspase-9 antibody, poly ADP-ribose polymerase (PARP) antibody (1: 1000; Cell Signaling Technology, Beverly, MA, USA), p38 mitogen-activated protein kinase (MAPK) antibody, p-p38 MAPK antibody (1: 500; KeyGen, Nanjing, China), or MKK3 antibody (1: 300; BOSTER) overnight at 4°C.

Techniques: Activation Assay, In Vitro, Western Blot, Negative Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Silencing Ras-Related C3 Botulinum Toxin Substrate 1 Inhibits Growth and Migration of Hypopharyngeal Squamous Cell Carcinoma via the P38 Mitogen-Activated Protein Kinase Signaling Pathway

doi: 10.12659/MSM.907468

Figure Lengend Snippet: Rac1 silencing inhibits the activation of P38 MAPK signal in vivo . The levels of MKK3 and p-MKK3 ( A, B ) and P38 and p-P38 ( C, D ) in each group were detected by Western blot analysis with GAPDH as the internal reference. All experiments were repeated 3 times. The results are presented as mean ±SD. N=6. *** P<0.001 compared with the negative control group.

Article Snippet: The membranes were blocked with 5% skim milk or 1% bovine serum albumin, and then incubated with Rac1 antibody, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1: 1000; Proteintech, Wuhan, China), Cyclin D1 antibody (1: 1000; BOSTER, Wuhan, China), Cyclin E antibody, Cyclin B antibody (1: 500; Bioss, Beijing, China), metal matrix proteinase (MMP)-2 antibody, MMP-9 antibody, B cell lymphoma-2 (Bcl-2) antibody, Bcl-2-associated X protein (Bax) antibody, phosphorylated-MAP kinase (p-MKK3) antibody (1: 500; Sangon Biotech, Shanghai, China), caspase-3 antibody, caspase-9 antibody, poly ADP-ribose polymerase (PARP) antibody (1: 1000; Cell Signaling Technology, Beverly, MA, USA), p38 mitogen-activated protein kinase (MAPK) antibody, p-p38 MAPK antibody (1: 500; KeyGen, Nanjing, China), or MKK3 antibody (1: 300; BOSTER) overnight at 4°C.

Techniques: Activation Assay, In Vivo, Western Blot, Negative Control