Flk 1

Frozen muscle tissue (~5–10 mg; 1:40, w/v) was homogenized as described before (Ballak et al. 2015 (link)). Homogenates were centrifuged at 10,000 g for 10 min at 4 °C. The supernatant was immediately stored at −80 °C and the protein concentration was measured using the DC protein assay kid (Bio-Rad Laboratories, Nazareth, Belgium). Forty micrograms of protein was separated by SDS-PAGE (10–12 % gels) and transferred to PVDF membranes. Membranes were blocked with 5 % nonfat milk for 1 h and then incubated overnight (4 °C) with the following antibodies: SDH (1:10,000, Santa Cruz, UK), cytochrome c oxidase subunit 4 (COX-4) (1:1000, Abcam, Cambridge, UK), Flk-1 (1:500, Santa Cruz, UK), and eukaryotic elongation factor 2 (eEF2) (1:1000, Cell Signaling Technology, Leiden, The Netherlands). Horseradish peroxidase-conjugated anti-mouse (1:10,000) or anti-rabbit (1:5000) secondary antibodies (Sigma-Aldrich, Bornem, Belgium) were used for chemiluminescent detection. Membranes were scanned and quantified with Genetools and Genesnap software (Syngene, Cambridge, UK), respectively. Preliminary experiments showed that eEF2 content was stable across the different treatments and conditions, and results are presented as the ratio protein of interest/eEF2.

After treatment, cells were harvested at the indicated times in lysis buffer (20 mmol/liter Tris (pH 7.5), 150 mmol/liter NaCl, 2 mmol/liter EDTA, 10% glycerol, 1% Triton X-100, 1 mmol/liter phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Sigma)). Protein concentrations were determined via BCA (Pierce, Rockford, IL), and proteins were resolved on 12% sodium dodecyl sulfate- (SDS-) polyacrylamide gels followed by electrophoretic transfer onto nitrocellulose membranes. The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline, 0.1% Tween 20, for 2 h and incubated at 4°C overnight with primary antibodies against TIAR (C-18) (1 : 500), Flk-1 (1 : 1000), Sox17 (1 : 500), VE-cadherin (1 : 1000), and c-myc (1 : 1000) (Santa Cruz Biotechnology, Santa Cruz, CA), nestin (1 : 500, Chemicon, Temecula, CA), and TIA-1 (1 : 500, Proteintech Group). Following incubation with appropriate horseradish peroxidase-conjugated secondary antibodies, signals were detected with an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ).

Formalin fixed E18.5 lungs were paraffin embedded and sectioned according to standard procedures. Paraffin sections were stained using antibodies specific to FOXF1 (Malin et al., 2007 (link)), FLK1 (Santa Cruz Biotechnology, Dallas, TX, USA), proSPC (Ustiyan et al., 2012 (link)) and α-SMA (Abcam) as described (Ustiyan et al., 2009 (link); Wang et al., 2010 (link)).

The protein expression levels of VEGF, FLK1, and GAPDH in the femoral head tissues obtained from rats in different groups were detected by western blot analysis. The protocol and semiquantitative analysis were carried out following the protocol of our previous study [26 (link)]. The following antibodies were used: VEGF antibody (rabbit antibody, dilution 1 : 50, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), FLK1 (rabbit antibody, dilution 1 : 50, Santa Cruz), and GAPDH antibody (internal control, rabbit polyclonal antibody, dilution 1 : 200, Santa Cruz).

The process was carried out in accordance with our previous study [21 (link)]. The anti-eNOS, PECAM-1, Flk-1, calponin, smooth muscle actin, transgelin-2, P2X1, P2X3, P2X5, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11 and P2Y14 antibodies were purchased from Santa Cruz Biotechnology. Protein extracts from the cell lines HMEC-1, C2 and MG-63 cells were applied as positive controls for the Western blot. Protein density was analyzed by Image J software (ImageJ 1.50i, Wayne Rasband National Institutes of Health, Bethesda, .MD USA) for P2 expression after differentiation.

Aortic rings extending microvessels were fixed with 4% formalin, permeabilised with 0.25% Triton-X for 15 min and incubated in PBS (+) containing 10% goat serum for 1 h at room temperature to block non-specific binding.After being rinsed with PBS (+) for 5 min they were then incubated overnight with primary antibody diluted in PBLEC29 (link). After rinsing with PBS (+) (3 × 10 min), samples were then incubated for 30 min at 37 °C in a cocktail of Alexa Fluor (488, 555, or Cy5) conjugated with appropriate secondary antibodies in PBLEC. Samples were rinsed then incubated (1 min) with Hoechst33342 and mounted using Fluoromount (Diagnostic BioSystems). Images were recorded using a fluorescence microscope (OlympusX70 or Keyence BZ-9000). The following primary antibodies were used: laminin (Progen 10765), CD31 (BD Pharmingen 550274), Reck [RECK-F, polyclonal rabbit antibodies (Matsuzaki et al., in preparation) and 5B11D1212 (link)]; α-Sma (DAKO MO85), fibronectin (BD Biosciences, 610078), PDGFR-beta (Santa Cruz Biotechnology sc-432), Flk-1 (Santa Cruz Biotechnology sc-625), LAMA5 (Sigma-Aldrich SAB4501720) and Ki67 (Leica Biosystems NCL-Ki67p).