Journal: Cancer medicine

Article Title: Direct effect of dasatinib on signal transduction pathways associated with a rapid mobilization of cytotoxic lymphocytes.

doi: 10.1002/cam4.925

Figure Lengend Snippet: Figure 1. Flow cytometric analysis of each lymphocyte subset and expression of phosphorylated proteins. Representative data are shown. Lymphocyte fractions are classified according to surface antibodies including CD3, CD8, and CD56. Natural killer (NK) cells were defined as the CD3-CD56+ immunophenotype and cytotoxic T lymphocytes (CTLs) were CD3+ CD8+ (A). Cells were costained with phospho-specific antibodies, including antibodies targeting pJAK1, pJAK2, pSTAT1, pSTAT3, pERK, pJNK, pp38, and pAKT, and expression levels of the isotype control and phosphorylated proteins in NK cells are presented (B). The values for the isotype control and each phosphorylated protein are shown as the median fluorescence intensity (MFI).

Article Snippet: Fluorescein isothiocyanate (FITC)- labeled polyclonal rabbit IgG antibodies for phosphorylated proteins, including pJAK1 (Y1034, #3238R), pJAK2 (Y1007, #2485R), pSTAT1 (Y701, #1657R), pSTAT3 (Y705, #1658R), pERK (T202/Y204, #1646R), pJNK (T183/Y185, #1640R), pp38 (T180/Y182, #2210R), pAKT (Y315, #5193R), and isotype control (#0295P) were purchased from Bioss (Wobun, MA, USA).

Techniques: Expressing, Control, Fluorescence

Journal: Cancer medicine

Article Title: Direct effect of dasatinib on signal transduction pathways associated with a rapid mobilization of cytotoxic lymphocytes.

doi: 10.1002/cam4.925

Figure Lengend Snippet: Figure 3. Constitutive levels of phosphorylated proteins including pJAK1, pJAK2, pSTAT1, pSTAT3, pERK, pJNK, pp38, and pAKT in natural killer (NK) cells (A) and cytotoxic T lymphocytes (CTLs) (B) grouped according to treatment (dasatinib [n = 18] or other TKI [n = 12]) are shown. The values for phosphorylated proteins in each fraction are shown as the median fluorescence intensity (MFI).

Article Snippet: Fluorescein isothiocyanate (FITC)- labeled polyclonal rabbit IgG antibodies for phosphorylated proteins, including pJAK1 (Y1034, #3238R), pJAK2 (Y1007, #2485R), pSTAT1 (Y701, #1657R), pSTAT3 (Y705, #1658R), pERK (T202/Y204, #1646R), pJNK (T183/Y185, #1640R), pp38 (T180/Y182, #2210R), pAKT (Y315, #5193R), and isotype control (#0295P) were purchased from Bioss (Wobun, MA, USA).

Techniques: Fluorescence

Journal: Drug Design, Development and Therapy

Article Title: Sitagliptin attenuates cardiomyopathy by modulating the JAK/STAT signaling pathway in experimental diabetic rats

doi: 10.2147/dddt.s109287

Figure Lengend Snippet: Figure 8 Effect of sitagliptin on STAT3 phosphorylation in the heart of control and diabetic rats. Notes: Photomicrographs of pSTAT3-immunostained heart sections of (A) control rats, (B) sitagliptin-treated rats showing the absence of immune positivity especially from the nuclei (arrow), (C) diabetic rats showing strong immune reactivity of almost all of myocardium cell nuclei and the endocardium endothelium cells (arrows), and (D) sitagliptin-treated diabetic rats showing many immunopositive nuclei of both endocardium endothelium and myocardium cells (arrows). Magnification ×400.

Article Snippet: Rabbit polyclonal anti-phospho-JAK2 and goat anti-phospho-STAT3 (pSTAT3) were supplied by Santa Cruz Biotechnology Inc. (Dallas, TX, USA).

Techniques: Phospho-proteomics, Control

Journal: PLoS ONE

Article Title: Acetylcholine Acts on Androgen Receptor to Promote the Migration and Invasion but Inhibit the Apoptosis of Human Hepatocarcinoma

doi: 10.1371/journal.pone.0061678

Figure Lengend Snippet: (A–D) Western blots showed the effects of Ach and MEC on STAT3 phosphorylation (A, B) and Akt phosphorylation (C, D) and in SNU-449 cells. Phosphorylation of STAT3 (A) and Akt (C) was enhanced by Ach in SNU-449 cells, whereas acetylcholine receptor antagonists MEC blocked the effect of Ach on STAT3 (B) and AKT (D) phosphorylation.

Article Snippet: Primary antibodies used here included rabbit monoclonal anti-AR (D6F11) (3H8; Cell Signaling Technology, Danvers, MA), rabbit monoclonal anti-nicotinic acetylcholine receptor alpha 7 (ab10096, abcam, USA), rabbit monoclonal anti-pSTAT3, anti-pAKT (195-14; BioCheck, Foster City, CA), and monoclonal anti-β-actin (Sigma).

Techniques: Western Blot

Journal: Oncogene

Article Title: The broad-range cyclin-dependent kinase inhibitor UCN-01 induces apoptosis in colon carcinoma cells through transcriptional suppression of the Bcl-x(L) protein.

doi: 10.1038/sj.onc.1207842

Figure Lengend Snippet: Figure 6 Variable response of NF-kB to UCN-01 treatment. The transcriptional activity of NF-kB is suppressed by UCN-01 treatment (1 mM, 48 h) in SW48 cell line, but it is activated in WiDr, HT-29 and LS513 cells Figure 7 UCN-01 treatment can inhibit both Ser727 and Tyr705 phosphorylation of STAT3 a. (a) Constitutive STAT3 phosphor- ylation at Ser727 is detectable in all cell lines. It is inhibited by UCN-01 (1 mM, 48 h) only in the apoptosis-performing cell lines. (b) STAT3 phosphorylation at Tyr705 is not detectable in any of the cell lines (shown LS513). It is inducible by IFNa and is inhibited within 1.5 h by UCN-01 treatment

Article Snippet: The following antibodies were employed: rabbit anti-BAX antibody (N20), goat anti-Akt1 antibody (C-20), mouse monoclonal anti-phosphotyrosine STAT3 antibody (clone B-7) (all from Santa Cruz Biotechnology, Heidelberg, Germany); mouse monoclonal anti-PARP antibody (clone C2-10), and mouse monoclonal anti-Bcl-xL (clone 2H12) (all from BD Pharmingen, Heidelberg, Germany); mouse monoclonal anti-b-actin antibody (clone AC-15) (Sigma-Aldrich Chemie, Taufkirchen, Germany).

Techniques: Activity Assay, Phospho-proteomics

Journal: Oncogene

Article Title: The broad-range cyclin-dependent kinase inhibitor UCN-01 induces apoptosis in colon carcinoma cells through transcriptional suppression of the Bcl-x(L) protein.

doi: 10.1038/sj.onc.1207842

Figure Lengend Snippet: Figure 8 Activation of STAT3 does not lead to Bcl-xL transcrip- tion. (a) Exogenous STAT3 is phosphorylated at Tyr705 after stimulation of SW48 cells with IL-6. (b) IL-6 stimulation activates the transcriptional activity of STAT3 as demonstrated by the increased activity of the luciferase reporter. (c) Bcl-xL transcription is not affected by the transcriptionally active STAT3. Total RNA was isolated from the same cells as used in (b), and the semiquantitative RT–PCR was carried out with the cycle number in the linear range of the amplification. b-actin was amplified for the control of the amounts of mRNA used in each lane

Article Snippet: The following antibodies were employed: rabbit anti-BAX antibody (N20), goat anti-Akt1 antibody (C-20), mouse monoclonal anti-phosphotyrosine STAT3 antibody (clone B-7) (all from Santa Cruz Biotechnology, Heidelberg, Germany); mouse monoclonal anti-PARP antibody (clone C2-10), and mouse monoclonal anti-Bcl-xL (clone 2H12) (all from BD Pharmingen, Heidelberg, Germany); mouse monoclonal anti-b-actin antibody (clone AC-15) (Sigma-Aldrich Chemie, Taufkirchen, Germany).

Techniques: Activation Assay, Activity Assay, Luciferase, Isolation, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Molecular nutrition & food research

Article Title: Modulation of Nrf2/Keap1 system by Wasabi 6-methylthiohexyl isothiocyanate in ARE-mediated NQO1 expression.

doi: 10.1002/mnfr.201200689

Figure Lengend Snippet: Figure 1. 6-MTITC increases the levels of NQO1 in a time (A) and dose-depended manner (B). HepG2 cells were treated with 10 M of 6-MTITC for 1.5–12 h or with 5–20 M of 6-MTITC for 6 h. Whole cell lysates were used to detect NQO1 and -tubulin by Western blot analysis with their respective antibodies. Densitometry of the blots was performed using the LumiVision Imager software. Histograms show the densitometric analysis of NQO1 normalized to -tubulin. Average value from triplicate tests with different superscript letters differs significantly at p < 0.05.

Article Snippet: The antibodies against Nrf2 (C-20), Keap1 (E-20), ubiquitin (P4D1), -tubulin (B-7), lamin B (C-20), rabbit IgG, and horseradish peroxidase conjugated anti-goat secondary antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Software

Journal: Molecular nutrition & food research

Article Title: Modulation of Nrf2/Keap1 system by Wasabi 6-methylthiohexyl isothiocyanate in ARE-mediated NQO1 expression.

doi: 10.1002/mnfr.201200689

Figure Lengend Snippet: Figure 5. 6-MTITC increases stabilization of Nrf2. (A) 6-MTITC in- creases Nrf2 level but decreases Keap1 level. HepG2 cells were treated with 0 M of 6-MTITC for 1.5–12 h and Nrf2, Keap1, and -tubulin were detected by Western blot analysis with their re- spective antibodies. Histograms show the densitometric analysis of Nrf2 comparing with control. Average value from triplicate tests with different superscript letters differs significantly at p < 0.05. (B) 6-MTITC extends Nrf2 stability. HepG2 cells were treated with 5 g/mL CHX or pretreated with 10 M 6-MTITC for 1.5 h and then treated with CHX for the indicated times. Nrf2, Keap1, and -tubulin were detected by Western blot analysis with their respective antibodies. Histograms show the densitometric analy- sis of Nrf2 comparing with control. Average value from triplicate tests with differently lettered superscripts differs significantly at p < 0.05.

Article Snippet: The antibodies against Nrf2 (C-20), Keap1 (E-20), ubiquitin (P4D1), -tubulin (B-7), lamin B (C-20), rabbit IgG, and horseradish peroxidase conjugated anti-goat secondary antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Control

Journal: Molecular nutrition & food research

Article Title: Modulation of Nrf2/Keap1 system by Wasabi 6-methylthiohexyl isothiocyanate in ARE-mediated NQO1 expression.

doi: 10.1002/mnfr.201200689

Figure Lengend Snippet: Figure 6. Effects of 6-MTITC on Nrf2 ubi- quitination and Keap1 modification. (A) Western blot analysis of endogenous Nrf2, Keap1, and ubiquitin. HepG2 cells were treated with 10 M of MG132 for 1 h and then treated with or without 10 M of 6-MTITC for 6 h. Whole-cell lysates were used to detect Nrf2, Keap1, and ubiquitin with their antibodies. Densitom- etry of the blots was performed using the LumiVision Imager software. Histograms show the densitometric analysis of ubi- quitin normalized to -tubulin. (B) and (C) Effects of 6-MTITC on ubiquitination of Nrf2 and Keap1. Equivalent amounts of proteins were immunoprecipitated with Nrf2 or Keap2 antibody and visualized by Western blot analysis with ubiquitin anti- body. The marker shows the ubiquitinated protein. Histograms show the densitomet- ric analysis of the ubiquitinated Nrf2. Aver- age value from triplicate tests with differ- ent superscript letters differs significantly at p < 0.05. (D) Modification of Keap1 by 6-MTITC. HepG2 cells were treated with 10 M of 6-MTITC for 6 h and analyzed by Western blot analysis with Keap1 anti- body. Densitometry of the blots was per- formed using the LumiVision Imager soft- ware. Histograms show the densitomet- ric analysis of modified Keap1 normalized to -tubulin. Average value from triplicate tests with differently lettered superscripts differs significantly at p < 0.05.

Article Snippet: The antibodies against Nrf2 (C-20), Keap1 (E-20), ubiquitin (P4D1), -tubulin (B-7), lamin B (C-20), rabbit IgG, and horseradish peroxidase conjugated anti-goat secondary antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Western Blot, Ubiquitin Proteomics, Software, Immunoprecipitation, Marker

Journal: Molecular nutrition & food research

Article Title: Modulation of Nrf2/Keap1 system by Wasabi 6-methylthiohexyl isothiocyanate in ARE-mediated NQO1 expression.

doi: 10.1002/mnfr.201200689

Figure Lengend Snippet: Figure 7. Effects of 6-MTITC on ARE activity in Nrf2- or Keap1- deficient cells. (A) Western blot analysis. HepG2 cells were trans- fected with 100 nM siRNA against Nrf2 or scrambled duplex in Opti-MEM culture medium for 5 h. The cells were further placed in DMEM medium for 24 h and then stimulated with 10 M of 6-MTITC for an additional 6 h. Whole cell lysates were used to de- tect Nrf2, NQO1, and -tubulin by Western blot analysis with their respective antibodies. Densitometry of the blots was performed using the LumiVision Imager software. Histograms show the den- sitometric analysis of Nrf2 or NQO1 normalized to -tubulin. Av- erage value from triplicate tests with different superscript letters (small for Nrf2 and capital for NQO1 in Fig. 7B) differs significantly at p < 0.05. (B) Reporter gene assay. HepG2 cells were cotrans- fected with pGL2-ARE luciferase construct, pGL4-TK-Renilla, and 100 nM siRNA against Nrf2 or scrambled duplex in Opti-MEM culture medium for 12 h. The cells were then placed in DMEM medium for 24 h and then stimulated by 10 M of 6-MTITC for an additional 12 h. The cells were lysed and assayed for firefly and Renilla luciferase activities. Average value from triplicate tests with differently lettered superscripts differs significantly at p < 0.05.

Article Snippet: The antibodies against Nrf2 (C-20), Keap1 (E-20), ubiquitin (P4D1), -tubulin (B-7), lamin B (C-20), rabbit IgG, and horseradish peroxidase conjugated anti-goat secondary antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activity Assay, Western Blot, Software, Reporter Gene Assay, Luciferase, Construct