2.8. Western Blotting and Measurement of the Phosphorylated-ERK Expression (p-ERK1/2)

Here, the same five samples of conditioned media used in the angiogenesis assay above were applied to BAEC pre-cultured for 48 h in serum poor (0.5% FBS) DMEM again using FGF-2 as a positive control and after 8 min incubation with an MFAT conditioned medium from the 24 h and five day samples at 37 °C. The following protocol was followed.

After rapid washing with ice-cold PBS, cells were lysed with an ice-cold radioimmunoprecipitation (RIPA) buffer (pH 7.5) containing 25 mM Tris-HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 0.5% SDS, 1 mM EDTA, 1 mM sodium orthovanadate (EGTA), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1% Triton × 100 and 1 μM leupeptin. The protein concentration of cell lysates was determined using the Bradford protein assay (Bio-rad, Watford, UK) and equal quantities of proteins (30 µg) were mixed with a 2 × Laemmli sample buffer boiled in a water bath for 15 min then centrifuged. The samples were separated along with pre-stained molecular weight markers (32,000–200,000 kDa) by 10% SDS-PAGE. Proteins were electro-transferred (Hoefer, Bucks, UK) onto nitrocellulose filters (Whatman, Protran BA85, Germany) and the filters were blocked for 1 h at room temperature in TBS-Tween (pH 7.4) containing 1% BSA. The membrane was then probed with phospho-ERK1/2 (Abcam, Cambridge, UK) diluted in the blocking buffer as indicated and incubated overnight at 4 °C on a rotating shaker. After washing (5 × 10 min in TBS-Tween at room temperature), the membrane was stained with either goat anti-rabbit or rabbit anti-mouse horse radish peroxidase (HRP) conjugated secondary antibodies diluted in TBS-Tween containing 5% de-fatted milk (1:2000, 1 h, room temperature) with continuous mixing. After a further five washes in TBS-Tween, proteins were visualised using enhanced chemi-luminescent detection (ECL, Thermo scientific, Cambridge, UK) and semi-quantitatively identified fold differences compared with house-keeping controls (α-tubulin, Abcam, Cambridge, UK) determined using Image-Lab software. All experiments were repeated at least twice, and a representative example is shown.