Journal: The FASEB Journal

Article Title: Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction

doi: 10.1096/fj.13-244210

Figure Lengend Snippet: CMV results in activated phosphorylation of STAT3 on Tyr705 and diaphragm contractile dysfunction. Diaphragms from mechanically ventilated (MV) rats and unfed controls were analyzed. A ) Ex vivo force–frequency relationship from mechanically ventilated ( n =13) rats and unfed controls ( n =7). B ) Western blots for total STAT3 and phospho-STAT3 Y705 from a representative set of mechanically ventilated rats and unfed controls (1, 3, 6, and 9 h, n =5–6 rats/group; 18 h, n =6–8). C ) Messenger RNA levels of the STAT3 downstream target genes SOCS3 and Myf5 from mechanically ventilated ( n =9) rats and unfed controls ( n =10). Mechanical ventilation period of 18 h. Results are means ± sem . * P < 0.05; Student's t test.

Article Snippet: Proteins electroblotted onto PVDF membranes were incubated with the following primary antibodies and the appropriate secondary antibodies: phospho-STAT3 [9131; Tyr705; Cell Signaling Technology (CST), Danvers, MA, USA], STAT3 (9132; CST), pSTAT3-S727 (NB2-12965; Novus Biologicals, Littleton, CO, USA), phospho-p38 (9215; Thr180/Tyr182; CST), myogenin (ab124800; Abcam, Cambridge, MA, USA), Ac-Histone H3 (9649; CST), tubulin (5346; CST), GRIM-19 [sc-136431; Santa Cruz Biotechnology (SCB), Dallas, TX, USA], 4-hydroxynonenal (4-HNE; ab46545; Abcam), lactate dehydrogenase A (LDHA; 2012S; CST), proliferating cell nuclear antigen (PCNA; sc-56; SCB), voltage-dependent anion channel (VDAC; 4661; CST), cytochrome c (4272; CST), caspase-3 (9664; CST), calpain 1 (2556; CST), and α-spectrin (sc-48382; SCB).

Techniques: Phospho-proteomics, Ex Vivo, Western Blot

Journal: The FASEB Journal

Article Title: Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction

doi: 10.1096/fj.13-244210

Figure Lengend Snippet: CMV increases Ser705 phosphorylation and mitochondrial accumulation of phospho-STAT3 (Ser727 and/or Tyr705/Ser727) within diaphragm muscle. A ) Diaphragms from unfed control rats ( n =8), MV-R548 rats ( n =9), or MV-Veh rats ( n =9) were analyzed by Western blot of total muscle lysate (representative animals depicted in blot) and quantitation. Correlation between increases in phospho-STAT3 S727 and phospho-STAT3 Y705 were determined. B ) Purified mitochondrial fractions (mito) and total lysates (total) were subjected to Western blot analysis for pSTAT3 S727 , pSTAT3 Y705 , total STAT3, GRIM-19, VDAC (mitochondrial marker), LDHA (cytoplasmic marker) and PCNA (nuclear marker). Unfed (U) controls, n = 10; MV-Veh (MV), n = 6; MV-R548, n = 7. Results are means ± sem . Mechanical ventilation period of 18 h. P values calculated by 1-way ANOVA with Tukey's post hoc analysis.

Article Snippet: Proteins electroblotted onto PVDF membranes were incubated with the following primary antibodies and the appropriate secondary antibodies: phospho-STAT3 [9131; Tyr705; Cell Signaling Technology (CST), Danvers, MA, USA], STAT3 (9132; CST), pSTAT3-S727 (NB2-12965; Novus Biologicals, Littleton, CO, USA), phospho-p38 (9215; Thr180/Tyr182; CST), myogenin (ab124800; Abcam, Cambridge, MA, USA), Ac-Histone H3 (9649; CST), tubulin (5346; CST), GRIM-19 [sc-136431; Santa Cruz Biotechnology (SCB), Dallas, TX, USA], 4-hydroxynonenal (4-HNE; ab46545; Abcam), lactate dehydrogenase A (LDHA; 2012S; CST), proliferating cell nuclear antigen (PCNA; sc-56; SCB), voltage-dependent anion channel (VDAC; 4661; CST), cytochrome c (4272; CST), caspase-3 (9664; CST), calpain 1 (2556; CST), and α-spectrin (sc-48382; SCB).

Techniques: Phospho-proteomics, Control, Western Blot, Quantitation Assay, Purification, Marker

Journal: The FASEB Journal

Article Title: Inhibition of Janus kinase signaling during controlled mechanical ventilation prevents ventilation-induced diaphragm dysfunction

doi: 10.1096/fj.13-244210

Figure Lengend Snippet: Proposed model for role of JAK signaling in VIDD. JAK signaling is activated by CMV and functions as a critical triggering mechanism upstream of STAT3 phosphorylation (Tyr705 and Ser705), mitochondrial dysfunction, ROS production, atrophy, and muscle weakness. Mechanical ventilation results in the mitochondrial accumulation of phospho-STAT3 (singly phosphorylated on Ser727 or doubly phosphorylated at both Ser727 and Tyr705). Import into mitochondria is facilitated through interaction of phospho-STAT3 with GRIM-19, a component of complex I of the ETC, and may directly affect mitochondrial function and ROS generation. Induction of various myogenic transcription factors and muscle-specific E3 ubiquitin ligases (MURF-1 and atrogin-1) and activation of calpain, caspase 9, and caspase 3 can contribute to muscle atrophy and proteolytic cleavage of myofilament proteins. Mitochondrial dysfunction may also lead to ROS-mediated modification of myofilament proteins in a manner that negatively affects contractile function.

Article Snippet: Proteins electroblotted onto PVDF membranes were incubated with the following primary antibodies and the appropriate secondary antibodies: phospho-STAT3 [9131; Tyr705; Cell Signaling Technology (CST), Danvers, MA, USA], STAT3 (9132; CST), pSTAT3-S727 (NB2-12965; Novus Biologicals, Littleton, CO, USA), phospho-p38 (9215; Thr180/Tyr182; CST), myogenin (ab124800; Abcam, Cambridge, MA, USA), Ac-Histone H3 (9649; CST), tubulin (5346; CST), GRIM-19 [sc-136431; Santa Cruz Biotechnology (SCB), Dallas, TX, USA], 4-hydroxynonenal (4-HNE; ab46545; Abcam), lactate dehydrogenase A (LDHA; 2012S; CST), proliferating cell nuclear antigen (PCNA; sc-56; SCB), voltage-dependent anion channel (VDAC; 4661; CST), cytochrome c (4272; CST), caspase-3 (9664; CST), calpain 1 (2556; CST), and α-spectrin (sc-48382; SCB).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Activation Assay, Modification

Journal: Clinical Cancer Research

Article Title: Reciprocal Regulation of c-Src and STAT3 in Non-Small Cell Lung Cancer

doi: 10.1158/1078-0432.ccr-09-0767

Figure Lengend Snippet: Fig. 4. Phosphorylated c-Src, STAT3, and downstream Src targets were measured in paired normal lung and NSCLC tumor tissues from patients who had previously undergone resection. A, tumors had higher mean c-Src activity, as indicated by decreased levels of inactive c-Src (pSrc Y527). Conversely, mean activated STAT3 (pSTAT3 Y705) was lower in tumor tissue than in normal lung. B, levels of inactive c-Src (pSrc Y527) correlated directly with activated STAT3 (pSTAT3 Y705) when analyzed as total levels of phosphorylated protein or ratio of phosphorylated protein to total protein. C, levels of phosphorylated FAK, p130Cas, and paxillin were inversely correlated with pSTAT3 Y705.

Article Snippet: Antibodies used in the Western blot analysis included c-Src and pSTAT3 S727 (Santa Cruz Biotechnology); pSrc Y419, pSTAT3 Y705, STAT3, pFAK Y861, Lyn, Yes, Bcl-XL, survivin, and STAT5 (Cell Signaling Technology); and β-actin (Sigma Chemical Company).

Techniques: Activity Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IL-4 and IL-13 guide early thymic progenitors to mature towards dendritic cells

doi: 10.4049/jimmunol.1701186

Figure Lengend Snippet: (A) Thymic cells from HR+/+ IL-13Rα1-GFP reporter mice were depleted of Lin+ cells and the Lin-CD4-CD8- cells were stained with antibodies to CD25, CD44, c-Kit, and intracellular pSTAT6(Y641), pSTAT1(Y701), pSTAT1(S727), pSTAT3 (S727) and pSTAT3 (Y705). The CD25-CD44+c-Kit+GFP+ (HR+ETPs) and CD25-CD44+c-Kit+GFP- (HR-/PETPs) were then analysed ex vivo for phosphorylation of STAT6, STAT1, and STAT3 at the indicated aa residues in comparison to isotype control for the corresponding anti-STAT antibody. The histograms show a representative experiment and the bar graphs show the mean ± SD MFI results compiled from 3 independent experiments. **p<0.01 as determined by two-tailed, unpaired Student’s t-test. (B, C) Thymic cells from HR−/− mice were depleted of Lin+ cells and the Lin-CD4-CD8- cells were stained with antibodies to CD25, CD44 and c-Kit, sorted as CD25-CD44+c-Kit+HR−/− ETPs, and transduced with HR-RV to drive HR expression. The cells were stained with anti-Thy1.1 and anti-IL-13Rα1 (marker for HR expression) and sorted as IL-13Rα1+Thy1.1+ cells. HR-RV transduced ETPs were cultured in the presence of 10ng/ml IL-4 alone (IL-4) or with STAT1 (IL-4 +STAT1 I) or STAT6 (IL-4 + STAT6 I) inhibitor. Similar culture conditions were used with 20 ng/ml IL-13 alone (IL-13) or with STAT1 (IL-13 + STAT1 I) or STAT6 (IL-13 + STAT6 I) inhibitor. Culture without cytokine or inhibitor (NIL) was included for control purposes. (B) STAT1 and ( C) STAT6 phosphorylation was analyzed after 1 hour incubation by flow cytometry. The histograms show representative experiments for STAT1 and STAT6 phosphorylation while the bar graphs show the mean ± SD MFI results compiled from three independent experiments. *p<0.05, **p<0.01 as determined by one-way ANOVA.

Article Snippet: Anti-CD3 (145–2C11), anti-CD4 (RM4–5), anti-CD8 (53–6.7), anti-CD25 (7D4), anti-CD44 (IM7), anti-CD45 (30-F11), anti-Pax5 (1H9), anti-CD45.1 (A20), anti-CD11b (M1/70), anti-CD11c (HL3), anti-CD117 (2B8), anti-IFNγR1 (XMG1.2), anti-pSTAT6 Y641 (J71–773.58.11), anti-pSTAT3 S727 (49/p-Stat3), anti-pSTAT3 Y705 (4/P-STAT3), anti-Zbtb46 (U4–1374), anti-SIRPα (P84), and anti-CD90.1 (OX-7), were purchased from BD biosciences (San Jose, CA).

Techniques: Staining, Ex Vivo, Two Tailed Test, Transduction, Expressing, Marker, Cell Culture, Incubation, Flow Cytometry