Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: The Journal of Experimental Medicine

Article Title: Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice

doi: 10.1084/jem.20062340

Figure Lengend Snippet: eNOS localization and activation in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries. (A and B) eNOS (A and B, green), Cav-1 (A, red), and GM130 (B, red) expression in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries in situ by whole-mount immunostaining. Bar, 100 nm. Nuclei (blue) are detected via DAPI labeling (C) caveolins, eNOS, HSP90; Akt protein expression and eNOS (Ser 1179 ) and Akt (Ser 473 ) phosphorylation in WT, Cav-1 KO, and Cav-1 RC pulmonary arteries. These blots are representative of four individual experiments. (D) Summary of eNOS and Akt phosphorylation-isolated vessels. Results are shown as phosphoprotein/total protein for eNOS and Akt, respectively. *, P, < 0.05, compared with WT vessels; †, P < 0.05, compared with Cav-1 KO vessels. Data are expressed as the mean ± the SEM.

Article Snippet: The membranes were probed with rabbit anti–Cav-1 antibody (BD Transduction Laboratories), mouse anti–Cav-2 antibody (BD Transduction Laboratories), mouse anti–Cav-3 antibody (BD Transduction Laboratories), mouse anti–β-actin antibody (Sigma-Aldrich), mouse anti-eNOS antibody (BD Transduction Laboratories), rabbit anti–phospho-eNOS (Ser 1179 ) antibody (BD Transduction Laboratories), mouse anti-HSP90 antibody (BD Transduction Laboratories), rabbit anti-Akt antibody (Cell Signaling Technology), rabbit anti–phospho-Akt (Ser 473 ) antibody (Cell Signaling Technology), rabbit anti-p42/44 antibody (Cell Signaling Technology), rabbit anti–phospho-p42/44 (Thr 202 /Tyr 204 ) antibody (Cell Signaling Technology), rabbit anti-p38 antibody (Cell Signaling Technology), rabbit anti–phospho-p38 (Thr 180 /Tyr 182 ) antibody (Cell Signaling Technology), rabbit anti-JNK antibody (Cell Signaling Technology), and rabbit anti–phospho-JNK (Thr 183 /Tyr 185 ) antibody (Cell Signaling Technology) at dilutions ranging from 1:200 to 1:1,000.

Techniques: Activation Assay, Expressing, In Situ, Immunostaining, Labeling, Isolation

Journal: The Journal of Experimental Medicine

Article Title: Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice

doi: 10.1084/jem.20062340

Figure Lengend Snippet: Enhanced Akt and p42/44 ERK phosphorylation and fibrogenic gene expression in hearts and lungs of WT, Cav-1 KO, and Cav-1 RC mice. (A and B) Total caveolins, eNOS, HSP90, Akt, p42/44, p38, and JNK protein expression and phosphor-Akt (Ser 473 ), p42/44 (Thr 202 /Tyr 204 ), p38 (Thr 180 /Tyr 182 ) in tissue extracts. (A) Typical Western blots from six individual experiments; (B) the summary of these experiments by quantitative imaging. Akt (Ser 473 ) phosphorylation (C) and p42/44 (Thr 202 /Tyr 204 ) phosphorylation (D) in main coronary arteries of the three strains of mice, respectively. Akt (Ser 473 ) phosphorylation (E) and p42/44 (Thr 202 /Tyr 204 ) phosphorylation (F) in pulmonary arteries (top) and lung parenchyma (middle showing large airway and bottom showing alveoli). Photos are representative from four separate experiments. Quantitative analysis of PECAM-1 and E-cad (G) and total nuclei (H) in hearts and lungs of WT, Cav-1 KO, and Cav-1 RC mice. Expression of TGF-β1, and interstitial collagen genes (I) by RT-PCR (compared with GAPDH) in total RNA isolated from hearts and lungs of WT, Cav-1 KO, and Cav-1 RC mice. Data are the means ± the SEM. n = 4 mice per group.

Article Snippet: The membranes were probed with rabbit anti–Cav-1 antibody (BD Transduction Laboratories), mouse anti–Cav-2 antibody (BD Transduction Laboratories), mouse anti–Cav-3 antibody (BD Transduction Laboratories), mouse anti–β-actin antibody (Sigma-Aldrich), mouse anti-eNOS antibody (BD Transduction Laboratories), rabbit anti–phospho-eNOS (Ser 1179 ) antibody (BD Transduction Laboratories), mouse anti-HSP90 antibody (BD Transduction Laboratories), rabbit anti-Akt antibody (Cell Signaling Technology), rabbit anti–phospho-Akt (Ser 473 ) antibody (Cell Signaling Technology), rabbit anti-p42/44 antibody (Cell Signaling Technology), rabbit anti–phospho-p42/44 (Thr 202 /Tyr 204 ) antibody (Cell Signaling Technology), rabbit anti-p38 antibody (Cell Signaling Technology), rabbit anti–phospho-p38 (Thr 180 /Tyr 182 ) antibody (Cell Signaling Technology), rabbit anti-JNK antibody (Cell Signaling Technology), and rabbit anti–phospho-JNK (Thr 183 /Tyr 185 ) antibody (Cell Signaling Technology) at dilutions ranging from 1:200 to 1:1,000.

Techniques: Expressing, Western Blot, Imaging, Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: Journal of Oncology

Article Title: High Aldehyde Dehydrogenase Levels Are Detectable in the Serum of Patients with Lung Cancer and May Be Exploited as Screening Biomarkers

doi: 10.1155/2019/8970645

Figure Lengend Snippet: Western blot analysis of the polyclonal anti-RTNKEASI serum in A549 cell line. SDS gel was loaded with protein extract from non-small cell lung cancer A549 cell line followed by electrophoresis and immunoblotting. The blots were incubated with the preimmune serum and the polyclonal anti-RTNKEASI serum produced in rabbits from Rockland (a) or BioGenes (b). The 55 kDa target of anti-RTNKEASI appears only in sera of animals after immunization, and it is absent in the presera from the same rabbit. These results were confirmed with both Rockland and BioGenes antibodies. A PCA was performed by incubating the corresponding blots with the polyclonal anti-RTNKEASI serum from Rockland, pretreated with or without the peptide RTNKEASI (c) and also β -actin, as endogenous control. The intensity of the band approximately at 55 kDa detected by the secondary anti-rabbit horseradish peroxidase-conjugated was reduced after the preincubation of the antibody with the peptide RTNKEASI.

Article Snippet: The production of two different rabbit polyclonal anti-RTNKEASI immune sera and the synthesis of TLP-derived peptide RTNKEASI [ ] were both conducted at Rockland Immunochemicals Inc. (Gilbertsville, PA, USA) and at BioGenes GmbH (Berlin, Germany).

Techniques: Western Blot, SDS-Gel, Electrophoresis, Incubation, Produced, Control

Journal: Journal of Oncology

Article Title: High Aldehyde Dehydrogenase Levels Are Detectable in the Serum of Patients with Lung Cancer and May Be Exploited as Screening Biomarkers

doi: 10.1155/2019/8970645

Figure Lengend Snippet: Western blot analysis for the polyclonal anti-RTNKEASI serum in cancer cell lines and cellular supernatant. (a) Protein extracts from non-small cell lung cancer A549, normal lung tissue MRC-5, Burkitt lymphoma CA46, leukemia HL60, breast cancer MCF-7, cervical carcinoma Hela, and prostate cancer PC-3 cell lines were loaded into SDS gel and subjected to electrophoresis and immunoblotting with the polyclonal anti-RTNKEASI serum from Rockland. High 55 kDa protein levels were identified in A549 cells and a lower amount in Hela, PC-3, CA46 and HL-60 cell lines whereas no protein levels were observed in MCF-7 cell line from breast cancer and in MRC-5 from normal lung tissue. An anti-ß-actin antibody was used for a loading control. (b) Protein extracts from cellular supernatant of non-small cell lung cancer A549 and cell lysate from A549 and MRC-5 cells were loaded into SDS gel and subjected to electrophoresis and immunoblotting with the polyclonal anti-RTNKEASI serum from Rockland. A specific band at 55 kDa was detected in the cellular supernatant sample with respect to cell lysate from A549; MRC-5 was used as negative control.

Article Snippet: The production of two different rabbit polyclonal anti-RTNKEASI immune sera and the synthesis of TLP-derived peptide RTNKEASI [ ] were both conducted at Rockland Immunochemicals Inc. (Gilbertsville, PA, USA) and at BioGenes GmbH (Berlin, Germany).

Techniques: Western Blot, SDS-Gel, Electrophoresis, Control, Negative Control

Journal: Journal of Oncology

Article Title: High Aldehyde Dehydrogenase Levels Are Detectable in the Serum of Patients with Lung Cancer and May Be Exploited as Screening Biomarkers

doi: 10.1155/2019/8970645

Figure Lengend Snippet: 2D gel electrophoresis and western blot analysis with cell lysates from A549 and MCF-7 cell lines using the polyclonal anti-RTNKEASI serum and preimmune serum. Protein extract from A549 and MCF-7 cells were loaded in 2D gel followed by electrophoresis and transferred to PVDF for immunoblotting with the polyclonal anti-RTNKEASI serum or preserum. A rectangle marks the location of the ALDHs detected with the polyclonal anti-RTNKEASI serum in A549 cells; the reactive spots indicated with arrows were absent in MCF-7 cells and in the blot incubated with the preserum. These protein spots at 55 kDa, excited and subjected to MS analysis, correspond to ALDH1A1.

Article Snippet: The production of two different rabbit polyclonal anti-RTNKEASI immune sera and the synthesis of TLP-derived peptide RTNKEASI [ ] were both conducted at Rockland Immunochemicals Inc. (Gilbertsville, PA, USA) and at BioGenes GmbH (Berlin, Germany).

Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot, Incubation