Anti pser473 akt

Cells were fixed with 4% PFA for 20 min at room temperature, or with methanol for 10 min at −20 °C, then cyto-centrifuged onto coverslips. Fixed cells were permeabilized and blocked for 30 min in PBS supplemented with 0.1% saponin and 3% bovine serum albumin, and then incubated with a primary and a secondary antibody for 1 h each. To stain with anti-Akt(pSer473) (Cell Signaling Technology; 193H12), 10% skimmed milk was used for blocking. After washing with PBS, cells were mounted with Fluomount (DiagnosticBioSystems). For staining endocytic compartments, cells were incubated for 1 h with 5 μg ml⁻¹ AF647-CTXB or 1 mg ml⁻¹ AF647-dextran (Molecular Probes). Confocal images were obtained by a Fluoview FV10i laser scanning microscope with an x60 1.20 N.A. water-immersion objective (Olympus). Composite figures were prepared with Photoshop elements 10 and Illustrator CS6 software (Adobe). Pearson’s correlation coefficients (Pearson’s R) were calculated with NIH ImageJ Version 1.48v software.

Rabbit anti-mouse lyve-1 antibody (RDI-103PA50) was from Research Diagnostics Incorporated (Concord, MA). Donkey anti-rabbit and rat IgGs conjugated with alexa 488, 594 were from TebuBio (TebuBio, Le Perray en Yvelines, France). Anti-pSer473Akt, anti-Akt, anti-pErk, anti-Erk1/2 are from Cell Signalling Technology. Anti-Phalloidin was from Cytoskeleton (actin-stain phalloidin 488, cat PHDG1). Anti-phospho-VEGFR-3 is from Cell applications Inc. Rabbit anti-human LC3B, anti-human Notch/NCID and anti-p62 were from Cell Signaling (#2775), Goat anti-human VE-Cadherin from Santa-Cruz ((C19):SC6458) and Rabbit anti-human ATG5 and ATG7 from Sigma. Anti-human active beta-catenin was from Milliopore.

The ipsilateral cortex tissue of the mouse brain was homogenized in prechilled buffer containing 50 mM Tris-HCl (pH 7.4), 2.0 mM EGTA, 2 mM Na3VO4, 50 mM NaF, 0.5 mM AEBSF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 4 μg/ml pepstatin A. Protein concentrations of the homogenates were determined by using Pierce 660 nm Protein Assay kit (Thermo Fisher Scientific Inc.). The samples were resolved in 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto PVDF membrane (Millipore, Bedford, MA). After blocking with 5% fat-free milk, the blots were then probed with a primary antibody, such as VEGF (1 : 1000; Abcam, USA), anti-AKT (1 : 1000; Cell Signaling Technology, USA), anti-pSer473-AKT (1 : 1000; Cell Signaling Technology, USA), anti-pSer9-GSK3β (1 : 1000; Cell Signaling Technology, USA), anti-GSK3β (1 : 1000; Cell Signaling Technology, USA), or anti-GAPDH (1 : 2000; Sigma, USA),washed and then incubated with a corresponding HRP-conjugated secondary antibody. The protein-antibody complex was visualized by using the Pierce ECL Western Blotting Substrate (Thermo Scientific) and exposed to a HyBlot CL autoradiography film (Denville Scientific, Inc. Metuchen, NJ). Specific immunostaining was quantified by using the Multi Gauge software V3.0 (Fuji Photo Film Co. Ltd.).

Female and male syndecan-4^−/− and WT isolated LV cardiomyocytes were fixed in 4% paraformaldehyde for 10 min, quenched in 150 mM glycine for 10 min and permeabilized with 0.5% Triton X-100 for 10 min, at room temperature. Cells were then plated on laminin coated (#L2020, mouse, Sigma Merck) glass bottom dishes (No 1.5, Ø 14 mm, γ-irradiated, Martek Corporation, United States). For visualization, cells were incubated with anti-pSer473-Akt (1:50, #9271, Cell signaling) overnight at 4°C, followed by secondary antibody labeling using goat anti-rabbit Alexa Fluor 647 plus (1:200, #32733, Thermo Fisher Scientific) for 1 h at room temperature. Wheat germ agglutinin (WGA) 488 (1:400, #W11261, Invitrogen) and DAPI 405 (0.1 mg/ml, #MBD0015, Sigma Aldrich) was included in the secondary step to visualize the sarcolemma and nuclei, respectively. Both primary and secondary antibodies were diluted in a blocking buffer consisting of 2% goat serum, 0.1% Triton X-100 and 0.02% NaN₃ in PBS. Imaging was performed on a ZEISS LSM 800 with Airyscan using the Airyscan super-resolution mode, using a plan-apo 63X 1.4 NA oil objective.