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Western Blot is used to detect a specific protein in a cell or tissue homogenate (or extract) [214]. The principle of the method lies in the separation of denaturated proteins, usually depending on their size (i.e., the length of the polypeptide chain), using gel electrophoresis and polyacrylamide gels under denaturating conditions. Initially, the protein extract is denaturated and solubilized by SDS (sodium dodecylsulfate). Typically, SDS-PAGE (polyacrylamide gel-electrophoresis) is used to separate the proteins. It maintains them in a denaturated state preventing formation of secondary and tertiary structures. In addition, the negatively charged SDS causes the proteins to adopt an overall negative net charge and thus allows their separation during electrophoresis, where they migrate toward the positive anode. Smaller proteins migrate faster through the polyacrylamide gel. Following, the proteins are electrophoretically transferred to a positively charged nitrocellulose membrane, causing irreversible binding. During transfer, the membrane is placed on the anode side of a transfer unit, resulting into a “pulling” of the negatively charged antigens onto the membrane. To prevent unspecific binding of antibodies during the subsequent stages, the membrane is blocked by a diluted protein solution – usually non-fat dry milk powder - that blocks binding sides on the membrane where no antigens are bound. The specific antigen of interest is detected by a primary antibody that in turn is recognized by a secondary antibody directed against the species- specific part of the primary antibody. The secondary antibody itself is conjugated to a reporter enzyme such as horseradish peroxidase that catalyzes a detectable

chemiluminscent reaction visualized on X-ray films. Alternatively, the secondary antibody can carry a radioactive label for detection purposes.

For Western Blots in this project, protein lysates were generated from embryonic EPCs using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce Biotechnology), according to the manufacturer’s instructions. In brief, cytoplasmic contents are released by disruption of the cell membranes. In a next step, the intact nuclei are recovered from the cytoplasmic extract by centrifugation, and nuclear proteins are extracted through lysis of the nuclei. The use of pure nuclear extract without the presence of cytoplasmic components elevates the quality to study interactions of DNA and nuclear proteins. To assure the stability of the nuclear extract, a proteinase inhibitor cocktail tablet (Roche Diagnostics, Indianapolis, IN, USA) was added to the extraction reagents (provided by Pierce Biotechnology) and the nuclear and cytoplasmic extracts were stored at -80ºC for further applications.

The total protein concentrations of the cell lysates were determined using the BCA Protein Assay Reagent Kit (Pierce Biotechnology). This assay allows colorimetric quantification of protein amounts by the reduction of Cu2+ to Cu+ by chelation of cupric ions through proteins in an alkaline environment (i.e. the biuret reaction or assay) [215]. The light blue to violet chelated complex absorbs light at 540 nm and the intensity of the colored signal is in proportion to the amount of peptides in the mixture, with four to six peptide bonds forming a complex with one cupric ion. For Western Blot, 3 µg of protein lysate were incubated together with 4 µl of 5X loading buffer (250 mM Tris-HCl pH 6.8, 500 mM DTT, 10% SDS, 20% Glycerin, 2% Bromphenol blue) in a total volume of 20 µl for 10 min at 70ºC to unfold the polypeptide chains, followed by electrophoresis using NUPage 4-12% Bis-Tris polyacryamide gels (Invitrogen) and a vertical gel electrophoresis unit (Hoefer Pharmacia Biotech Inc., San Francisco, CA, USA) at 85 V for 2 hours in running buffer (24 mM Tris-Base, 192 mM glycin, 3.4 mM SDS, pH 7.4) Next to the protein samples, a molecular weight ladder (SeeBlue Plus2 Prestained Standard - Invitrogen) was run. Following electrophoresis, a Hybond-ECL nitrocellulose membrane (Amersham Biosciences) was placed onto the gel and the complex was covered on both sides with blotting paper (VWR International, West Chester, PA, USA) and a transfer sponge on the outside (VWR). The whole complex was soaked in transfer buffer (48 mM Tris-Base, 39 mM glycine, 0.038% SDS, 20% methanol

pH 8 - Sigma-Aldrich) and placed into a transfer unit (Hoefer), followed by electrophoretic blotting at 60 V for 2 hours in transfer buffer. Subsequently, the membrane was incubated for 1 hour in 5% non-fat dry milk powder (Nestle Corporation) at room temperature under shaking at 100 rpm, followed by incubation with the primary antibody for 12 hours at 4ºC or 1 hour at room temperature. The membrane was incubated with goat polyclonal anti-Foxc1 or goat polyclonal anti- Foxc2 (Abcam Incorporation, Cambridge, MA, USA – both at a concentration of 0.5 µg/ml, diluted in 5% non-fat dry milk powder) or mouse monoclonal anti-β-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Following incubation, the membrane was washed 5x with PBST buffer (PBS, 0.05% Tween20 - Sigma-Aldrich) for 10 min each and subsequently incubated with a secondary antibody (0.2 µg/ml in 5% non-fat milk powder) directed against the Fc part of mouse or goat antibodies and

conjugated with horseradish peroxidase (Santa Cruz). After incubation for 2 h at room temperature under shaking at 100 rpm, the membrane was washed 5x with PBST buffer, followed by the development of chemiluminescent light reaction, using the ECL Western Blotting Analysis System (Amersham). This kit provides the substrate reagent and buffers for the horseradish peroxidase enzyme and allows to develop a light reaction that was subsequently detected using a X-ray film (CL-X Posure Film, Pierce Biotechnology).

Horseradish peroxidase catalyzes the oxidation of chemiluminescent substrates such as luminol in the presence of H2O2 [216], and this enzymatic activity is then

visualized on X-ray films.