Journal: Circulation Research

Article Title: BMPER Is an Endothelial Cell Regulator and Controls Bone Morphogenetic Protein-4–Dependent Angiogenesis

doi: 10.1161/circresaha.108.178434

Figure Lengend Snippet: Figure 1. BMPER expression, localization, and regulation by FoxO3a in endothelial cells. A, Expression of BMPER in human vascular endothelial cells of different origin. Cells were lysed and subjected to Western blot analysis with the indicated antibodies. B and C, Localization of BMPER was characterized by immuno- cytochemistry in C166 mouse yolk sac endothelial cells. Corre- sponding serum was used as negative control. Nuclei were stained with DAPI. Scale bar100 m. D and E, Silencing of FoxO3a in HUVECs with 2 different siRNAs compared to scram- bled siRNA control resulted in enhanced BMPER expression shown by RT-PCR (D) and Western blot analysis (E). Seventy- two hours after transfection, mRNA expression was analyzed by using specific primers for FoxO3a, BMPER, and human RNA polymerase II. Western blot analysis was performed with the indicated antibodies. -Tubulin served as loading control. Rep- resentative Western blots are shown, along with densitometric analysis of the time course of BMPER expression. F, BMPER mRNA expression at 72 and 96 hours after transfection with FoxO3awt or the constitutively active mutant FoxO3aA3 com- pared to empty vector. BMPER mRNA was quantified by real- time (quantitative) PCR using specific primers for BMPER and hRP as internal control. BMPER mRNA expression was calcu- lated using the CT method. MeansSD. *P0.05 vs control.

Article Snippet: Total RNA was extracted from HUVEC using the Aurum RNA Mini Kit (Bio-Rad).

Techniques: Expressing, Western Blot, Immunocytochemistry, Negative Control, Staining, Control, Reverse Transcription Polymerase Chain Reaction, Transfection, Mutagenesis, Plasmid Preparation, Real-time Polymerase Chain Reaction

Journal: Circulation Research

Article Title: BMPER Is an Endothelial Cell Regulator and Controls Bone Morphogenetic Protein-4–Dependent Angiogenesis

doi: 10.1161/circresaha.108.178434

Figure Lengend Snippet: Figure 2. Specific silencing of BMPER by siRNA in HUVECs. A, BMPER mRNA expression after 24 and 48 hours posttrans- fection with the siRNA BMPER I and II, respectively, compared to scrambled siRNA control. BMPER mRNA was quantified by real-time (quantitative) PCR using specific primers for BMPER and human RNA poly- merase II as internal control. Knockdown efficiency was calcu- lated using CT method. MeansSD; n4. *P0.001 vs con- trol. B, Representative semiquantitative RT-PCR analysis 24 hours posttransfection is shown. C, Western blot analysis was performed with the indicated antibodies 48 hours posttransfection.

Article Snippet: Total RNA was extracted from HUVEC using the Aurum RNA Mini Kit (Bio-Rad).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: The FASEB Journal

Article Title: Protease-activated receptor 2 activates airway apical membrane chloride permeability and increases ciliary beating

doi: 10.1096/fj.201700114RRR

Figure Lengend Snippet: Expression of PAR-2 in airway epithelial cells. A) PAR-2 expression in primary airway epithelial cell lines and primary cells from 3 patients was assayed by reverse transcription-PCR. Oral squamous epithelium cDNA was used as a control for PAR-4. b.p., base pair; Pt, patient; rt, reverse transcription. B) PAR-2 expression was confirmed by Western blot in primary sinonasal ALI culture samples (6 separate patients, unrelated to patients in A. C) Immunofluorescence using a PAR-2 antibody in submerged BEAS-2B cells revealed plasma membrane–localized staining. GLUT1 was a control for membrane localization. D, E) Immunofluorescence of PAR-2 in primary dissociated sinonasal ciliated cells showing colocalization with NKCC1 (D) and Na+K+ ATPase (E). A distinct gap was noted (arrows) between the base of the cilia (labeled with β-tubulin IV), corresponding to the apical cell body membrane and the start of basolateral NKCC1/Na+K+ ATPase immunofluorescence. PAR-2 rabbit pAb was used with mouse mAb anti-NKCC1 (top). PAR-2 mouse mAb was used with rabbit mAb anti-Na+K+ATPase (bottom). Scale bars, 10 µm. F) Representative scatter plots of PAR-2 intensity and either NKCC or Na+K+ ATPase. For all dissociated cells imaged (n = 15 with NKCC1 and n = 17 with Na+K+ ATPase), Pearson’s correlation coefficient (Pearson’s R) and Mander’s overlap coefficient (Mander’s R) for basolateral marker and PAR-2 were both ≥0.95.

Article Snippet: Human PAR-2 and PAR-4 samples were amplified by PCR using an iCycler (Bio-Rad, Hercules, CA, USA), RedTaq DNA Polymerase (MilliporeSigma), and the following primers: 5′-GCTGGTCACCATCCCTTTGT-3′ and 5′-TCTGCTTTACAGTGCGGACA-3′ (hPAR-2) and 5′-CCTGGTTTATCTCTACCGGCG-3′ and 5′-CTCTCGTCGTAGACGCTGGG-3′ (hPAR-4) from Integrated DNA Technologies (Coralville, IA, USA). cDNA was prepared from cultured cells using Trizol (Thermo Fisher Scientific) with rDNAse I (Ambion, Austin, TX, USA) and an iScript cDNA Synthesis Kit (Bio-Rad).

Techniques: Expressing, Reverse Transcription, Control, Western Blot, Immunofluorescence, Clinical Proteomics, Membrane, Staining, Labeling, Marker

Journal: Molecular Genetics & Genomic Medicine

Article Title: Clinical application of single‐molecule optical mapping to a multigeneration FSHD1 pedigree

doi: 10.1002/mgg3.565

Figure Lengend Snippet: Molecular diagnosis of FSHD1. (a) Southern blot hybridization of selected family members with FSHD1. Membrane‐bound EcoRI/HindIII (E/H), EcoRI/HindIII/BlnI (E/H/B), and HindIII (H) digested genomic DNA from affected and normal family members were hybridized to p13E‐11 (4q35 and 10q26), 4qA, and 4qB labeled probes. 4qA (*) and 4qB (+) fragments. (b) FISH analysis of family member 28‐III (FSHD1). Green signals represent D4Z4 repeat numbers. (c) SMOM analysis of FSHD1 family members. BssSI maps (vertical green bars) of 4qA (purple) and 4qB (orange) alleles for family members 28‐III and 58‐IV (FSHD1) and 59‐IV (normal). The position and number ( n ) of D4Z4 repeats for each allele are indicated by horizontal purple and orange bars

Article Snippet: The WBCs were resuspended in cell suspension buffer, embedded into agarose plugs (CHEF Genomic DNA Plug Kit, Bio‐Rad).

Techniques: Biomarker Discovery, Southern Blot, Hybridization, Membrane, Labeling

Journal: Arthritis and rheumatism

Article Title: Modulation of the angiogenic phenotype of normal and systemic sclerosis endothelial cells by gain-loss of function of pentraxin 3 and matrix metalloproteinase 12.

doi: 10.1002/art.27522

Figure Lengend Snippet: Figure 1. Effects of transient transfection of normal microvascular endothelial cells (N-MVECs) with matrix metalloproteinase 12 (MMP12) and pentraxin 3 (PTX3), in comparison with systemic sclerosis (SSc) MVECs, assessed as shape change (A), adhesion/spreading properties (B), and expression of target proteins (C). A, The morphologic profile of normal MVECs, either left untreated or following electroporation with the pCDNA3 vector alone or along with MMP12 or PTX3 complementary DNA, was compared with that of SSc MVECs. B, The percentage of spread cells in relation to adherent cells was assessed as a function of time under the following culture conditions: normal MVECs treated with vector alone (pCDNA3), with the vector carrying the PTX3 gene (pPTX3), or with the vector carrying the MMP12 gene (pMMP12), and untreated SSc MVECs. Bars show the mean SD results of 3 experiments performed in triplicate. C, Western blotting was used to assess MMP-12 and PTX3 protein expression in culture medium containing transfected normal MVECs incubated with anti-MMP12 or anti-PTX3 antibodies. Untreated normal MVECs under nontransfected control conditions; pCDNA3 normal MVECs electroporated with plasmid pCDNA3; pMMP12 or pPTX3 normal MVECs electroporated with plasmid pMMP12 or pPTX3. Numbers on the right indicate molecular weights. Representative results are shown from 1 of 3 experiments that yielded similar results.

Article Snippet: To produce the recombinant vector, 1 g of RNA was retrotranscribed using the iScript complementary DNA synthesis kit (Bio-Rad), followed by amplification by polymerase chain reaction (PCR) with specific primers: for MMP12, forward ATG-CGG-TAC-CAT-GGG-GAA-GTT-TCT-TCTAAT-A and reverse ATG-CCT-CGA-GCT-AAC-AAC-CAAACC-AGC-TAT-TGC-TTT, and for PTX3, forward ATGCGG-TAC-CGT-CAT-GGG-GCA-TCT-CCT-TGC-GATTCT and reverse TAC-GCT-CGA-GTG-AAA-CAT-ACTGAG-CTC-CTC-CA.

Techniques: Transfection, Comparison, Expressing, Electroporation, Plasmid Preparation, Western Blot, Incubation, Control

Journal: Pain

Article Title: MicroRNA-mediated GABA Aα-1 receptor subunit down-regulation in adult spinal cord following neonatal cystitis-induced chronic visceral pain in rats.

doi: 10.1016/j.pain.2012.09.002

Figure Lengend Snippet: Fig. 4. (A) Real-time RT-PCR analysis of differentially expressed GABAAa-1 gene in spinal dorsal horns (L6–S1) from rats with neonatal cystitis. Two different treatment protocols were followed. Tissues were examined on P60 from the unchallenged protocol and on P30 from the re-challenged protocol. The ordinate value 2DCT corresponds to the mRNA expression relative to reference gene ribosomal RNA (rRNA); data are represented as mean ± SD (n = 5 per group). (B) Western blot analysis of GABAAa-1 protein in spinal dorsal horn samples (L6–S1) from rats with neonatal cystitis. The intensity of GABAAa-1 immunoreactivity for different tissues was normalized against the intensity of b-actin expression for the same tissue. Results are expressed as means ± SD (n = 5 per group). ⁄Significant difference at p < 0.05. (C) GABAAa-1 immunostaining of differentially expressed GABAAa-1 gene in spinal dorsal horns (L6–S1) from rats with neonatal cystitis. The tissues were examined on P60 after zymosan/saline treatment at P14 to P16. The scale bar is 50 lm. The intensity of staining for 10 individual cells from each group was determined; data are presented as means ± SD. ⁄Significant difference at p < 0.001.

Article Snippet: GABAAa-1 gene expression in spinal dorsal horn neurons (L6–S1) by real-time quantitative RT-PCR Total RNA was extracted from spinal dorsal horn samples from both the unchallenged (P60) and re-challenged (P30) groups of animals using Total RNA extraction kit from Bio-Rad (#732-6830).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Immunostaining, Saline, Staining