Journal: PloS one

Article Title: Live-cell imaging of the association of STAT6-GFP with mitochondria.

doi: 10.1371/journal.pone.0055426

Figure Lengend Snippet: Figure 3. Immunoimaging assays showing colocalization of STAT6 with mitochondria in the cytoplasm of digitonin- washed Hep3B, HPAEC and HPASMC cells. The indicated cells were grown in 6-well plates for one day, washed 4x with the digitonin (50 mg/ml)-0.3M sucrose buffer and then fixed followed by immuno- fluoresence analyses as indicated using either the anti-STAT6 pAb (green) and F1-ATPase mAb (red). Insets are shown at high magnification within each panel. Scale bars = 10 mm. Quantitative colocalization analyses using the Pearson’s and Costes’ plugins in Image J confirmed colocalization between STAT6 and F1-ATPase immunofluorescence at a setting of P,0.05 in Panels A, B and C. doi:10.1371/journal.pone.0055426.g003

Article Snippet: PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55426 Antibodies and competing peptides Rabbit polyclonal antibodies to STAT6 (sc-621x), glucose-6phosphate dehydrogenase (sc-67165), the Flag tag (sc-807) and the respective relevant competing peptide to STAT6 (sc-621P) and the irrelevant OctA peptide (sc-835P) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Immunofluorescence

Journal: PloS one

Article Title: Live-cell imaging of the association of STAT6-GFP with mitochondria.

doi: 10.1371/journal.pone.0055426

Figure Lengend Snippet: Figure 4. Characterization of the specificityof the anti-STAT6 immunofluoresence and its organellar localization by immu- nogold electron microscopy. Panel A. Anti-STAT6 immunofluore- sence assayed using the rabbit pAb colocalizing with F1-ATPase in the cytoplasm of digitonin-washed Hep3B cells was compared to that of an irrelevant pAb (G6PD). Scale bar = 10 mm. Panel B. Peptide competition characterization of the anti-STAT6 pAb immunofluorescence using either the relevant STAT6 peptide that had been used as the immunogen or the irrelevant OctA peptide. Scale bar = 10 mm. Panel C. Immunogold EM localization of the anti-STAT6 pAb immunoreactiv- ity. Cryo-thin sections of HPAECs were probed using ant-STAT6 pAb and 18-nm-tagged anti-rabbit IgG as indicated in ref. 23. Insets are shown at high magnification. Scale bars = 500 nm. Similar data were obtained using Hep3B cells (not shown). doi:10.1371/journal.pone.0055426.g004

Article Snippet: PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55426 Antibodies and competing peptides Rabbit polyclonal antibodies to STAT6 (sc-621x), glucose-6phosphate dehydrogenase (sc-67165), the Flag tag (sc-807) and the respective relevant competing peptide to STAT6 (sc-621P) and the irrelevant OctA peptide (sc-835P) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Electron Microscopy, Immunofluorescence

Journal: PloS one

Article Title: Live-cell imaging of the association of STAT6-GFP with mitochondria.

doi: 10.1371/journal.pone.0055426

Figure Lengend Snippet: Figure 5. STAT6 peptides in Hep3B and STAT6SH2-/-SH2- MEF -derived cell fractions. Panel A. Western blot analyses of cell extracts (duplicate lanes) prepared from Hep3B cultures (90 mm plates) without and after washing 4x with the digitonin-sucrose buffer and the 7.5K mitochondria- enriched cell pellet derived from STAT6SH2-/-SH2- MEFs (rightmost single lane). The amount of cell extracts used in the Hep3B lanes corresponds to that derived from equal numbers of cells. Left and right blots show immunoblotting after competition with the unrelated OctA peptide or the relevant STAT6-peptide. Panel B. The 2x washed P7.5K pellet prepared from duplicate batches of Hep3B cultures (five confluent 90 mm cultures/group) were subjected to anti-TOM22-mAb magnetic bead immunoisolation chromatography. The flow-through (FT) and the Bound fractions were resedimented, checked microscopically for enrichment of mitochondria in the Bound fraction and then respective aliquots (20% of each fraction) used for Western blotting. The full-length STAT6 peptide is shown. Panel C. Hep3B cells (ten 90 mm cultures/group) were harvested and used to prepare the 2x washed P7.5K membrane pellet fraction. This was then sedimented through a Percoll (30%)-0.25 M sucrose gradient as indicated in ‘‘Materials and Methods,’’ the respective gradient fractions collected and prepared for Western blotting (half of each fraction for each blot) using the indicated probes. doi:10.1371/journal.pone.0055426.g005

Article Snippet: PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55426 Antibodies and competing peptides Rabbit polyclonal antibodies to STAT6 (sc-621x), glucose-6phosphate dehydrogenase (sc-67165), the Flag tag (sc-807) and the respective relevant competing peptide to STAT6 (sc-621P) and the irrelevant OctA peptide (sc-835P) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Derivative Assay, Western Blot, Chromatography, Membrane

Journal: PloS one

Article Title: Live-cell imaging of the association of STAT6-GFP with mitochondria.

doi: 10.1371/journal.pone.0055426

Figure Lengend Snippet: Figure 6. Targeting of STAT6-GFP to mitochondria in Hep3B cells assayed in live-cells using MitoTracker as the mitochon- drial vital stain. Panels A and B. Hep3B cells in 35–mm plates were transfected with the STAT6-GFP expression plasmid and 2 days later the cultures were exposed to MitoTracker Red CMXRos at 100 nM for 15 min. After washing with PBS the live cells were imaged using a 40x water-immersion objective in green (STAT6-GFP) and red (MitoTracker). Insets are shown at high magnification within each panel. Scale bars = 10 mm. Quantitative colocalization analyses using the Pearson’s and Costes’ plugins in Image J confirmed colocalization between STAT6-GFP and MitoTracker fluorescence at a setting of P,0.05 in Panels A and B. doi:10.1371/journal.pone.0055426.g006

Article Snippet: PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55426 Antibodies and competing peptides Rabbit polyclonal antibodies to STAT6 (sc-621x), glucose-6phosphate dehydrogenase (sc-67165), the Flag tag (sc-807) and the respective relevant competing peptide to STAT6 (sc-621P) and the irrelevant OctA peptide (sc-835P) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Staining, Transfection, Expressing, Plasmid Preparation, Fluorescence

Journal: PloS one

Article Title: Live-cell imaging of the association of STAT6-GFP with mitochondria.

doi: 10.1371/journal.pone.0055426

Figure Lengend Snippet: Figure 7. Targeting of STAT6-GFP but not of N1-GFP to mitochondria in Hep3B cells assayed in live-cells using TMRE as the mitochondrial vital stain. Hep3B cells in 35–mm plates were transfected with expression plasmids for N1-GFP (Panel A) or STAT6-GFP (Panel B) and 2 days later the cultures were exposed to TMRE at 5 nM for 15 min. After washing with PBS the live cells were imaged using a 40x water-immersion objective by two-color fluorescence in green (N1- GFP or STAT6-GFP) and red (TMRE). Insets are shown at high magnification within each panel. Scale bars = 10 mm except in Panel B, insets = 5 mm. doi:10.1371/journal.pone.0055426.g007

Article Snippet: PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55426 Antibodies and competing peptides Rabbit polyclonal antibodies to STAT6 (sc-621x), glucose-6phosphate dehydrogenase (sc-67165), the Flag tag (sc-807) and the respective relevant competing peptide to STAT6 (sc-621P) and the irrelevant OctA peptide (sc-835P) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Staining, Transfection, Expressing, Fluorescence

Journal: PloS one

Article Title: Live-cell imaging of the association of STAT6-GFP with mitochondria.

doi: 10.1371/journal.pone.0055426

Figure Lengend Snippet: Figure 8. Targeting of STAT61–459-GFP to mitochondria in Hep3B cells. Panels A and B. Hep3B cells in 35–mm plates were transfected with expression constructs for either the full-length or the 1-459 truncated version of STAT6-GFP. Two days later the plates were either fixed and immunostained for F1-ATPase (Panel A) or exposed to MitoTracker Red CMXRos at 100 nM for 15 min Panel B) and imaged as indicated in Materials and Methods. Scale bars = 10 mm. doi:10.1371/journal.pone.0055426.g008

Article Snippet: PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55426 Antibodies and competing peptides Rabbit polyclonal antibodies to STAT6 (sc-621x), glucose-6phosphate dehydrogenase (sc-67165), the Flag tag (sc-807) and the respective relevant competing peptide to STAT6 (sc-621P) and the irrelevant OctA peptide (sc-835P) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Transfection, Expressing, Construct

Journal: PloS one

Article Title: Live-cell imaging of the association of STAT6-GFP with mitochondria.

doi: 10.1371/journal.pone.0055426

Figure Lengend Snippet: Figure 9. STAT6 associated with mitochondria in MEFs derived from wildtype (wt) or STAT6SH2-/SH2- mice. Panel A. MEFs derived from wt and STAT6SH2-/SH2- grown in 35–mm plates were washed using the digitonin-sucrose buffer and then immunostained for STAT6 and F1-ATPase. The cells were imaged using a 100x oil immersion objective. Insets represent the boxed regions within each frame. Scale bar = 10 mm. Panels B and C. Whole-cell extracts of MEFs derived from wt or STAT6SH2-/SH2-mice were evaluated (each in duplicate lane per variable) using Western blotting without (Panel B), and with peptide-competition (Panel C). doi:10.1371/journal.pone.0055426.g009

Article Snippet: PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e55426 Antibodies and competing peptides Rabbit polyclonal antibodies to STAT6 (sc-621x), glucose-6phosphate dehydrogenase (sc-67165), the Flag tag (sc-807) and the respective relevant competing peptide to STAT6 (sc-621P) and the irrelevant OctA peptide (sc-835P) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).

Techniques: Derivative Assay, Western Blot

Journal: bioRxiv

Article Title: A porcine ex vivo lung perfusion model to investigate bacterial pathogenesis

doi: 10.1101/708503

Figure Lengend Snippet: A. CD163 surface expression in PBS or Kp52145-infected pBMDMs by flow cytometry. Values are shown as standard error of mean of two independent experiments in duplicate. **, p<0.01 determined by unpaired Student’s -test. B. Immunoblotting analysis of phosphorylation of STAT6 (PSTAT6) and tubulin in lysates of pBMDMs infected with Kp52145 for the indicated times or left uninfected (ni). Data is representative of three independent experiments. C. cd163 levels in pBMDMs non-infected (ni) or infected with Kp52145 pre-treated with STAT6 inhibitor (AS1517499, 50nM 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments. D. Arginase-1 levels in pBMDMs non-infected (ni) or infected with Kp52145 pre-treated with STAT6 inhibitor (AS 1517499, 50nM 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments. E. Immunoblotting analysis of phosphorylation of STAT6 (PSTAT6) and tubulin in lysates of pBMDMs infected with Kp52145 and the isogenic cps mutant, strain 52145-Δ wca K2 for the indicated times or left uninfected (ni). Data is representative of three independent experiments. F. cd163 levels in pBMDMs non-infected (ni) or infected with the cps mutant, strain 52145-Δ wca K2 , pre-treated with STAT6 inhibitor (AS1517499, 50nM 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments in duplicate. G. arginase-1 levels in pBMDMs non-infected (ni) or infected with cps mutant, strain 52145-Δ wca K2 , pre-treated with STAT6 inhibitor (AS 1517499, 50nM 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments in duplicate. In panels C, and D, ***p<0.001, **p<0.01, for the indicated comparisons using one-way ANOVA with Bonferroni correction.

Article Snippet: Primary antibodies included: phospho-STAT6 (Tyr641) (1:2000, #9361), total STAT6 (1:1000, BioRad #170-6516), phospho-STAT3 (Y705) (1:2000, #9145), total STAT3 (1:2000, #12640), phospho-ERK (p44/42) (1:2000; #91015), phopsho-p38 (T180/Y182) (1:2000, #4511), all from Cell Signalling Technologies.

Techniques: Expressing, Infection, Flow Cytometry, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: A porcine ex vivo lung perfusion model to investigate bacterial pathogenesis

doi: 10.1101/708503

Figure Lengend Snippet: A. il-10 levels in pBMDMs non-infected (ni) or infected with Kp52145 pre-treated with STAT6 inhibitor (AS 1517499, 50nM/ 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments. B. Immunoblotting analysis of phosphorylation of STAT3 (PSTAT3) and STAT3 in lysates of pBMDMs infected with Kp52145 for the indicated times or left uninfected (ni). Data is representative of three independent experiments. C. Immunoblotting analysis of phosphorylations of ERK (pERK), p38 (Pp38), and tubulin in lysates of pBMDMs infected with Kp52145 for the indicated times or left uninfected (ni). Data is representative of three independent experiments. D. il-10 levels in pBMDMs non-infected (ni) or infected with Kp52145 pre-treated with p38 inhibitor (SB203580, Tocris, 10 μg/mL, 2 h prior to infection), ERK inhibitor (U0126, LC laboratories, 20 μg/mL, 2 h prior to infection) or DMSO vehicle control. Values are shown as standard error of mean of three independent experiments. In panels A and D, ****p < 0.0001, ***p < 0.001, for the indicated comparisons using one-way ANOVA with Bonferroni correction.

Article Snippet: Primary antibodies included: phospho-STAT6 (Tyr641) (1:2000, #9361), total STAT6 (1:1000, BioRad #170-6516), phospho-STAT3 (Y705) (1:2000, #9145), total STAT3 (1:2000, #12640), phospho-ERK (p44/42) (1:2000; #91015), phopsho-p38 (T180/Y182) (1:2000, #4511), all from Cell Signalling Technologies.

Techniques: Infection, Western Blot