Journal: Biomedicines

Article Title: Bioartificial Cardiac Patches Functionalized with Apelin-13 Increase Cardiac C-Type Natriuretic Peptide Expression in Infarcted Rats

doi: 10.3390/biomedicines14020266

Figure Lengend Snippet: Representative phase contrast and immunofluorescence micrographs showing neovascularization within and under the patch. ( a ) Image showing the new blood vessel formation in the Infarct + A-13p group. Red arrows indicate the neovessels (less than 20 µm in diameter) within the patch. Scale Bar 100 μm. ( b ) Partial reconstruction of the anterior LV wall after α-SMA/TnC/DAPI immunofluorescence staining in the Infarct + A-13p group. On the right panels, a higher magnification of the area within the black box shows α-SMA staining. Red arrows indicate the neovessels (less than 20 µm in diameter) inside the cardiac tissue. Scale Bar 100 μm. α-SMA: green; TnC: orange; DAPI: blue; M: myocardium; P: patch.

Article Snippet: Tissue sections were then incubated with primary antibodies directed against anti-TnC (1:30, sc-48347, Santa Cruz, Germany) and anti-smooth muscle α-actin (α-SMA) (1:50, ab5694, Abcam, Cambridge, UK) O/N at 4 °C, followed by secondary antibodies conjugated to phycoerythrin (1:200, sc-3738, Santa Cruz, Heidelberg, Germany) and Alexa Fluor 488 (1:200, A11070, Invitrogen, Thermo Fisher, Monza, Italy), respectively, for 2 h at RT.

Techniques: Immunofluorescence, Staining

Journal: IUCrJ

Article Title: Structural and biochemical insights into Zn 2+ -bound EF-hand proteins, EFhd1 and EFhd2

doi: 10.1107/S2052252523001501

Figure Lengend Snippet: In vitro actin co-sedimentation assay (F-actin binding) and negative staining electron microscopy. SDS–PAGE analysis of in vitro actin co-sedimentation assays with EFhd1 and EFhd2. Protein samples (12 µ M ) were added to polymerized ( a ) β-actin (8 µ M ) or ( b ) α-actin (8 µ M ) in the presence of 1 m M EGTA, 1 m M CaCl 2 or 20 µM ZnCl 2 . ( c ) Co-sedimentation ratios from each experiment. Filled and open black squares show the co-sedimentation ratios for EFhd1 and EFhd2 with β-actin, respectively, while filled and open red spheres show the co-sedimentation ratios for EFhd1 and EFhd2 with α-actin, respectively. Symbols and error bars represent the mean and 95% confidence interval for the mean, which were calculated from five independent experiments. Negatively stained electron micrographs: ( d ) F-actin assembled from β-actin in the presence of EFhd1 and 1 m M EGTA, 20 µ M CaCl 2 or ZnCl 2 ; ( e ) F-actin assembled from α-actin in the presence of EFhd2 and 1 m M EGTA, 20 µ M CaCl 2 or ZnCl 2 .

Article Snippet: In brief, non-muscle actin (85% β-actin and 15% γ-actin) derived from human platelets and muscle actin (α-actin) derived from rabbit skeletal muscle (Cytoskeleton Inc.) were mixed in G-buffer [0.2 m M CaCl 2 , 5 m M Tris–HCl (pH 8.0)] to produce actin stock and were polymerized in an actin polymerization buffer [100 m M KCl, 2 m M MgCl 2 , 0.5 m M ATP, 0.2 m M Tris–HCl (pH 8.0)] for 1 h at 24°C.

Techniques: In Vitro, Sedimentation, Binding Assay, Negative Staining, Electron Microscopy, SDS Page, Staining

Journal: PLoS ONE

Article Title: Ablation of the Cardiac-Specific Gene Leucine-Rich Repeat Containing 10 ( Lrrc10 ) Results in Dilated Cardiomyopathy

doi: 10.1371/journal.pone.0051621

Figure Lengend Snippet: (A) LRRC10 cofractionates with α-actin and α-actinin in the particulate fraction. Heart extracts from 10-week-old mice were fractionated into a nonparticulate fraction (Non) and a particulate fraction (Par) enriched in myofibril proteins. GAPDH and myosin heavy chain (MyHC) were used as nonparticulate and particulate controls, respectively. (B) Yeast-two hybrid screening identified α-actinin and γ-actin as binding partners of LRRC10. (C) α-actinin and α-actin endogenously interact with LRRC10 in the heart. WT or Lrrc10 − / − mouse heart extracts were immunoprecipitated (IP) with preimmune serum (PreIM) or an LRRC10 antibody and immunblotted for α-actinin or α-actin. (D) LRRC10 interacts with α-, β-, and γ-actin in vitro. Purified α-actin or cytoskeletal actin was incubated with GST or a GST-LRRC10 fusion protein and pulled down proteins were immunoblotted for α-actin, γ-actin, or β-actin. (E) LRRC10 colocalizes with α-actin. Coimmunostaining for LRRC10 and α-actin in adult mouse ventricular cardiomyocytes. Enlargement of boxed area is shown at bottom . Scale bar = 10 μm.

Article Snippet: 5 μg of purified rabbit skeletal muscle α-actin (Cytoskeleton, AKL95) or 20 μg of cytoskeletal nonmuscle actin (Cytoskeleton, APHL95, 85% β-actin, 15% γ-actin) was incubated with 1.5 μg GST or GST-LRRC10 for two hours in incubation buffer (PBS, 0.1 mM CaCl 2 , and 1% Triton X-100) followed by washing in wash buffer (20 mM Tris HCl, pH 7.4 and 150 mM NaCl).

Techniques: Two Hybrid Screening, Binding Assay, Immunoprecipitation, In Vitro, Purification, Incubation