Journal: PLoS ONE

Article Title: Up-Regulation of hERG K + Channels by B-RAF

doi: 10.1371/journal.pone.0087457

Figure Lengend Snippet: A. Representative original western blot showing hERG membrane protein abundance (anti-K v 11.1 antibody, Alamone Labs) analyzed by cell surface biotinylation in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (Control) or with 10 µM B-RAF inhibitor PLX-4720 (PLX-4720). B. Arithmetic means ± SEM (n = 7, arbitrary units) of normalized hERG membrane protein abundance analyzed by cell surface biotinylation in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (white bar) or with 10 µM B-RAF inhibitor PLX-4720 (black bar). *(p<0.05) indicates statistically significant difference from rhabdomyosarcoma RD cells treated with vehicle alone. C. Representative original dot plots of hERG-FITC positive cells at the cell surface analysed by flow cytometry in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (Control) or with 10 µM B-RAF inhibitor PLX-4720 (PLX-4720); FL-1 Height: hERG-FITC fluorescence intensity. D. Arithmetic means ± SEM (n = 5, %) of normalized percentage of positive cells showing hERG expression at the cell surface analyzed by flow cytometry in rhabdomyosarcoma RD cells after 24 hours treatment with vehicle alone (white bar) or with 10 µM B-RAF inhibitor PLX-4720 (black bar). *(p<0.05) indicates statistically significant difference from rhabdomyosarcoma RD cells treated with vehicle alone.

Article Snippet: After blocking with 5% non-fat dry milk in TBS 0.1% Tween20 for 1 hour at RT, the blots were incubated overnight at 4°C with rabbit anti-K v 11.1 (hERG, extracellular) antibody (diluted 1:200, Alamone Labs, Jerusalem, Israel).

Techniques: Western Blot, Flow Cytometry, Fluorescence, Expressing

Journal: bioRxiv

Article Title: High Shear Stress Reduces ERG Causing Endothelial-Mesenchymal Transition and Pulmonary Arterial Hypertension

doi: 10.1101/2024.02.02.578526

Figure Lengend Snippet: (A) Confocal microscopic images of lung tissue sections from 4 patients with PAH and a congenital heart defect (APAH-CHD), 4 patients with Idiopathic or Familial PAH (I/FPAH, 2 each), or healthy controls (Donor, n=4). ERG (red), EC marker vWF (green) and nuclei stained with DAPI (blue). Arrows in magnified images (right column) point to EC with ERG in the nucleus. Scale bars, 40 μm in the three left columns, 15 μm in the magnified images. Quantification shows the average percentage of ERG-positive EC per small pulmonary artery (SPA) per patient/control, *p<0.05 vs Donor by One-way ANOVA and Kruskal-Wallis multiple comparisons test. (B-E) Human donor PAEC transfected with ERG siRNA (si ERG ) or nontargeting siRNA (siCON) followed by 48 h LSS (15 dyn/cm 2 ). ( B ) Representative immunoblot and densitometric analysis. ( C ) Representative immunofluorescent staining shown with intensity quantified in an average of 5 fields of view per sample. Decreasing ERG leads to reduced PECAM1 (top left), reduced CDH5 (lower left), and increased ACTA2 (Right). Scale bars, 50 μm. (D) BMPR2 mRNA by qRT-PCR. (E) Representative immunoblot and densitometric analysis of BMPR2. n=4 biological replicates, *p<0.05; **p<0.01; ***p<0.001 vs. siCON under LSS, by paired Student t test.

Article Snippet: Sections were blocked and permeabilized in blocking buffer (3% BSA plus 5% goat, 0.2% Triton X-100 in 1XTBS) for 1 h at RT followed by incubation with primary antibodies against ERG (Santa Cruz, 1:100) for 72 h and EC marker, vWF (Abcam, 1:250) for 48 h at 4°C.

Techniques: Marker, Staining, Control, Transfection, Western Blot, Quantitative RT-PCR