Journal: PLoS ONE

Article Title: NDRG2 Ameliorates Hepatic Fibrosis by Inhibiting the TGF-β1/Smad Pathway and Altering the MMP2/TIMP2 Ratio in Rats

doi: 10.1371/journal.pone.0027710

Figure Lengend Snippet: (A) Real-time PCR was used to detect the expression of Smad2, Smad3 and Smad7 in LX-2 cells with TGF-β1 and/or adenovirus treatment. (B) Western blotting was used to detect the expression of Smad2, phospho-Smad2, Smad3, phospho-Smad3 and Smad7 in LX-2 cells with TGF-β1 and/or adenovirus treatment. (C) Real-time PCR was used to detect the expression of MMP2 and TIMP2 in LX-2 cells following TGF-β1 and/or adenovirus treatment. β-actin was used as a loading control. All experiments were performed independently in triplicate. *P<0.05 compared with the TGF-β1 or TGF-β1+AdLacZ group.

Article Snippet: The blots were probed with the following primary antibodies: NDRG2 (Abnova), β-actin, Smad7 (Santa Cruz Biotechnology), α-SMA (Abcam), Smad2/phospho-Smad2 (pSmad2), Smad3/phospho-Smad3 (pSmad3) (Cell Signaling Technology), followed by incubation with species-matched secondary antibodies.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: PLoS ONE

Article Title: NDRG2 Ameliorates Hepatic Fibrosis by Inhibiting the TGF-β1/Smad Pathway and Altering the MMP2/TIMP2 Ratio in Rats

doi: 10.1371/journal.pone.0027710

Figure Lengend Snippet: (A) Real-time PCR was used to detect the expression of Smad2, Smad3 and Smad7 in rats with PBS or adenovirus treatment. (B) Western blotting was used to detect the expression of Smad2, phospho-Smad2, Smad3, phospho-Smad3 and Smad7 in rats with PBS or adenovirus treatment. (C) Real-time PCR was used to detect the expression of MMP2 and TIMP2 in rats following PBS or adenovirus treatment. β-actin was used as a loading control. “normal” means rats without treatment. *P<0.05 compared with the PBS or AdLacZ group.

Article Snippet: The blots were probed with the following primary antibodies: NDRG2 (Abnova), β-actin, Smad7 (Santa Cruz Biotechnology), α-SMA (Abcam), Smad2/phospho-Smad2 (pSmad2), Smad3/phospho-Smad3 (pSmad3) (Cell Signaling Technology), followed by incubation with species-matched secondary antibodies.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: PLoS ONE

Article Title: NDRG2 Ameliorates Hepatic Fibrosis by Inhibiting the TGF-β1/Smad Pathway and Altering the MMP2/TIMP2 Ratio in Rats

doi: 10.1371/journal.pone.0027710

Figure Lengend Snippet: Primers for Real-time PCR.

Article Snippet: The blots were probed with the following primary antibodies: NDRG2 (Abnova), β-actin, Smad7 (Santa Cruz Biotechnology), α-SMA (Abcam), Smad2/phospho-Smad2 (pSmad2), Smad3/phospho-Smad3 (pSmad3) (Cell Signaling Technology), followed by incubation with species-matched secondary antibodies.

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: p70 Ribosomal Protein S6 Kinase Is a Checkpoint of Human Hepatic Stellate Cell Activation and Liver Fibrosis in Mice

doi: 10.1016/j.jcmgh.2021.09.001

Figure Lengend Snippet: CEP-1347 does not inhibit phosphorylation of JNK, ERK1, ERK2, or SMAD2 but reduces phosphorylation of p70S6K in human HSCs (LX-2). Human LX-2 cells were stimulated with ( A ) TGF-β or ( B , C ) PDGF-BB and co-stimulated with 1000 nM CEP-1347 (CEP) for ( A ) 24 hours or ( B , C ) 1 hour. ( A ) Densitometry data of phosphorylated protein expression levels were normalized to that of unphosphorylated proteins. The results are illustrated as representative blots (at least 6 per group; ∗ P < .05; ∗∗ P < .01; analysis of variance). ( B ) Phosphorylation of p70S6K (pp70S6K) was assessed by immunomicroscopy (green) and compared with unphosphorylated p70S6K. (C) Protein expression of pp70S6K was quantified by densitometry. Densitometric values of p70S6K were normalized to unphosphorylated p70S6K (n = 13; ∗ P < .05; analysis of variance).

Article Snippet: Membranes were incubated with the primary antibodies directed against the following molecules: α-SMA (Sigma-Aldrich) PDGF receptor-β (Cell Signaling Technology, Danvers, MA), collagen 1α1 (R&D Systems, Minneapolis, MN), JNK, phospho-JNK (pJNK), ERK1/2, phospho-ERK1/2 (pERK), SMAD2, phospho-SMAD2 (pSMAD2), AKT, phospho-AKT (pAKT), p70S6K, phospho-p70S6K (pp70S6K) (all from Cell Signaling Technology), GAPDH (Abcam, Cambridge, United Kingdom) and β-actin (Sigma-Aldrich).

Techniques: Expressing

Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

Article Title: Pentabromopseudilin: a myosin V inhibitor suppresses TGF- β activity by recruiting the type II TGF- β receptor to lysosomal degradation

doi: 10.1080/14756366.2018.1465416

Figure Lengend Snippet: MyoVa depletion enhances T β RII turnover and mitigates TGF- β /Smad signalling. A549 cells were infected with the lentivirus carrying control (Ctr shRNA) or MyoVa shRNA constructs (MyoVa shRNA#1 and MyoVa shRNA#2). Three days post-infection, total protein was extracted and subjected to Western blot using anti-MyoVa, anti-T β RII or anti- β -actin antibodies. (A) Two MyoVa shRNAs abolish MyoVa protein production and reduced the T β RII protein levels. (B) Effects of MyoVa on TGF- β -induced Smad2/3 phosphorylation. A549 cells harbouring MyoVa shRNA#1 or control shRNA were stimulated with 5, 10, 20, 50 and 100 pM of TGF- β for 30 min. The amount of pSmad2/3 obtained from the lysates of cells in the absence (Lanes 8–12) or presence (Lanes 1–7) of MyoVa was analysed through Western blotting using anti-pSmad2/3, anti-Smad2/3 and anti- β -actin were used as loading control. (C) MyoVa depletion inhibits TGF- β -induced fibronectin, PAI-1, and N-cadherin expression. Control and MyoVa-silenced A549 cells were treated with 5, 10, 20, 50 and 100 pM of TGF- β for 48 h. The protein abundance of cells in the absence (Lanes 8–12) or presence (Lanes 1–7) of MyoVa was analysed through Western blotting using appropriate antibodies. Right graphs illustrate quantitative analyses of ECL (mean ± SD) from at least three independent experiments; ** p < .01.

Article Snippet: Rabbit polyclonal antibodies against pSmad2/3 (#8828), Smad2/3 (#8685), PAI-1 (#11907), Flag tag (#8146), Lamp-1 (sc-9091) and MyoVa (#3402) were purchased from Cell Signalling (Boston, MA).

Techniques: Infection, shRNA, Construct, Western Blot, Expressing

Journal: Respiratory Research

Article Title: Airway hyperresponsiveness is associated with airway remodeling but not inflammation in aging Cav1 -/- mice

doi: 10.1186/1465-9921-14-110

Figure Lengend Snippet: Alterations of the airway remodeling features in mice after OVA challenge in Cav1 -/- mice. Representative pictures of 1 week-OVA challenged WT and Cav1 -/- mouse lung sections. (A , B . ) Goblet cells metaplasia was detected using Periodic Acid Schiff staining. Positive cells (indicated by arrows) were counted in airways of 100-200 μm in an average of 5 airways per animals is reported (Bar graph). (C , D . ) Detection of α-SMA positive cells by immunohistochemistry and reporting the surface of positive staining relative to the length of basement membrane (Bar graph). (E , F . ) Detection of collagen depositon around the airways using Picrosirius red staining and quantification of the relative amount of collagen deposition (Bar graph). (G , H . ) Detection of pSmad2 using immunohistochemistry. Activation of the canonical TGF–β signaling pathway was also determined by western blot. Densitometric quantitations were carried out using the whole lung homogenates of WT and Cav1 -/- mice after a 1-week OVA challenge. Values are represented as relative protein levels of pSmad2 to total Smad2 (Bar graph). For all the measurement, * < p 0.05 represents significance by 2-way ANOVA between WT and Cav1 -/- mice for the same age group, and # < p 0.05 represents significance by 2-way ANOVA between same age group for WT and Cav1 -/- mice.

Article Snippet: Following routine techniques of deparaffinization, antigen retrieval and quenching of peroxidase activity, the slides were incubated with primary pSmad2/3 antibody (cat# 9510S, Cell signaling, Danvers, MA) at a 1:500 dilution for 12 h, then biotinylated secondary antibody from VectaStain Elite ABC Mouse kit (PK-6102, Vector Laboratories Burlingame, CA 94010).

Techniques: Staining, Immunohistochemistry, Activation Assay, Western Blot

Journal: PLoS ONE

Article Title: Transforming Growth Factor β and Insulin Signal Changes in Stromal Fibroblasts of Individual Keratoconus Patients

doi: 10.1371/journal.pone.0106556

Figure Lengend Snippet: Two DN and 6 KC keratocyte cultures were treated with TGF β1 (2 ng/ml for 30 minutes) and were immunoblotted for pSMAD2/3, pSMAD1/5/8 and GAPDH (A). Densitometry scans of relevant bands were normalized to GAPDH and compared by one-way ANOVA and * indicates p≤0.05 (B and C).

Article Snippet: The following primary antibodies were used: anti-pSMAD2/3 and anti-pSMAD1/5/8 (Cell Signaling Technology, Beverly, MA), anti-p21 (monoclonal IgG, BD Biosciences), and pAKT1-ser473 (#4060, Cell Signaling Technology) and anti-GAPDH (Santa Cruz, Dallas, TX).

Techniques: