Journal: Animals : an Open Access Journal from MDPI

Article Title: Effect of the TGF-β/BMP Signaling Pathway on the Proliferation of Yak Pulmonary Artery Smooth Muscle Cells under Hypoxic Conditions

doi: 10.3390/ani14142072

Figure Lengend Snippet: Impact of hypoxia on BMP/TGF-β signaling pathway-related protein expression and proliferation in yak PASMCs. ( A – C ) Western blot analysis of HIF-1α and HIF-2α protein expression under hypoxic conditions, with a densitometric analysis performed using ImageJ. ( D – G ) Western blot analysis of TGF-β/Smad2/3 signaling pathway-related protein expression under hypoxia conditions, with a densitometric analysis performed using ImageJ. ( H – J ) Western blot analysis of BMP/Smad1/5 signaling pathway-related protein expression under hypoxic conditions, with a densitometric analysis performed using ImageJ. ( K – M ) Western blot analysis of BCL-2 and PCNA protein expression levels under hypoxic conditions, with a densitometric analysis performed using ImageJ. ** p < 0.01, * 0.01 < p < 0.05, ns indicates insignificant difference.

Article Snippet: The membrane was blocked at room temperature with 50 g·L −1 non-fat dry milk powder for 3 h. Then, the samples were incubated overnight at 4 °C with primary antibodies for HIF-1α (1:1000; ab16066, Abcam, Cambridge, MA, USA), HIF-2α (1:800; AF6362, Affinity, Jiangsu, China), TGF-β1 (1:500; bs-0086R, Bioss, Beijing, China), Smad2 (1:500; bs-0718R, Bioss, Beijing, China), Smad3 (1:500; bs-8853R, Bioss, Beijing, China), phosphorylated Smad2 (P-Smad2) (1:400; bs-8853R, Bioss, Beijing, China), phosphorylated Smad3 (P-Smad3) (1:400; bs-3425R, Bioss, Beijing, China), BMPR2 (1:500, bs-4237R; Bioss, Beijing, China), Smad1/5 (1:500; AF0614, Affinity, Jiangsu, China), phosphorylated Smad1/5 (P-Smad1/5) (1:400; bs-3418R, Bioss, Beijing, China), PCNA (1:500; bs-2006R, Bioss, Beijing, China), and BCL-2 (1:500; bs-0032R, Bioss, Beijing, China).

Techniques: Expressing, Western Blot

Journal: The European journal of neuroscience

Article Title: Balance of pro-apoptotic transforming growth factor-beta and anti-apoptotic insulin effects in the control of cell death in the postnatal mouse retina.

doi: 10.1111/j.1460-9568.2005.04183.x

Figure Lengend Snippet: Fig. 6. Effect of transforming growth factor (TGF)-b and insulin on Smad levels and its nuclear translocation. (a) Western blots with a specific Smad1 ⁄ 2 ⁄ 3 antibody demonstrating a significant up-regulation of Smad 24 h after addition of exogenous TGF-b to the basal culture medium. In contrast, exogenous insulin down-regulates Smad levels. Double treatment with both factors likewise partially prevented the increase of endogenous Smad levels. (b) Quantification of the Western blot data shown in (a). Values are means ± SEM. *P < 0.05; **P < 0.01; calculated by one-way anova test and Newman-Keuls post-test comparing all experimental groups. (c) Immunocytochemical staining of cells cultured for 1 h in the presence of exogenous TGF-b, insulin or both factors revealed that TGF-b treatment results in a translocation of Smad to the nucleus of apoptotic cells (4–6), whereas the Smad staining in apoptotic cells remains cytoplasmic in the basal (1– 3) and the TGF-b plus insulin (7–9) groups. Arrows, pyknotic nuclei of apoptotic cells; small arrowheads, cytoplasmic Smad staining; large arrowheads, Smad translocation to the nucleus. Scale bar, 5 lm. DAPI, 4¢,6-diamidino-2-phenylindole.

Article Snippet: Membranes were blocked in phosphate-buffered saline with 0.05% Tween20 and 8% skim powered milk (or 8% bovine serum albumin for phospho-antibodies) for 2 h at room temperature (18 –25 C) and incubated overnight at 4 C with primary antibodies [TGF-b receptor (TbR)I, sc-398, 1 : 100, Santa Cruz; TbRII, sc-400, 1 : 100, Santa Cruz; Smad1 ⁄ 2 ⁄ 3, sc-7960, 1 : 100, Santa Cruz; a a 2005 Federation of European Neuroscience Societies, European Journal of Neuroscience, 22, 28–38 P-Bcl-2, 1 : 1,000, Cell Signalling; Bcl-2, 1 : 500, BD Biosciences, Heidelberg, Germany; Bcl-XL, 1 : 1000, New England Biolabs, Frankfurt, Germany] diluted in phosphate buffer with Tween20 and 5% skim powered milk (or 5% bovine serum albumin for phosphoantibodies).

Techniques: Translocation Assay, Western Blot, Staining, Cell Culture

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Inhibition of MicroRNA‐155 Supports Endothelial Tight Junction Integrity Following Oxygen‐Glucose Deprivation

doi: 10.1161/JAHA.118.009244

Figure Lengend Snippet: Efficiency of microRNA‐155 (miR‐155) inhibition and overexpression. A, Diagram describing the experimental setup. Human primary brain microvascular endothelial cells (HBMECs) seeded in cell culture inserts were subjected to 3 hours of oxygen‐glucose deprivation (OGD) and returned back to the normal cell culture conditions; 24 hours later, the cells were transfected with miR‐155 inhibitor, mimic, or appropriate scrambled oligonucleotide. Cells were analyzed at 48 hours after the transfection. B, Fluorescence confocal microscopy of HBMEC s transfected with fluorescein‐labeled miR‐155 inhibitor control (green dots) and stained for actin with rhodamine‐conjugated fluorescent phalloidin. Left panel: orthogonal image projection verifies that fluorescent probes were incorporated within the cell. Bar: 10 μm. C and D, miR‐155 PCR analysis. Total RNA was isolated from the cells subjected to OGD and transfected with the following oligonucleotides: miR‐155 mimic ( OGD /M; grey bar); mimic control ( OGD / MC; grey bar with white stripes); specific miR‐155 inhibitor ( OGD /I; black bar); and control inhibitor ( OGD / IC ; black bar with white stripes). C, P =0.002, (D) P =0.029, Mann–Whitney (Wilcoxon) test. n=3 (for OGD / MC and OGD / IC groups) and n=4 (for OGD /I and OGD /M groups) independent experiments. E, Validation of miR‐155 inhibition by the quantitative assessment of miR‐155 direct target proteins Rictor and SMAD ‐1. Optical density of the protein bands was measured using ImageJ software, normalized to GAPDH density in every sample, and expressed as average relative density values. Protein levels were compared between OGD /I (black bars) and OGD / IC (black bars with white stripes) cell lysates. P =0.029, Mann–Whitney (Wilcoxon) test, n=3 independent experiments per group. Representative immunoblots demonstrate Rictor and SMAD ‐1 protein expression (3 bands per group); GAPDH was used as a loading control. Error bars: SEM ; * P <0.05; ** P <0.01.

Article Snippet: SMAD‐1 and Rictor proteins were detected using SMAD proteins (Cat# 12656) and mTOR pathway (Cat# 9964) antibody sampler kits (Cell Signaling).

Techniques: Inhibition, Over Expression, Cell Culture, Transfection, Fluorescence, Confocal Microscopy, Labeling, Control, Staining, Isolation, MANN-WHITNEY, Biomarker Discovery, Software, Western Blot, Expressing