Gel documentation system

The two primers MF (5′-GAGAGCAAGCACCCTTTCCT-3′) and MR (5′-GAGAGTCCTGTGATTCCCCT-3′) were used to amplify the complete coding region of MC1R gene from the 108 individual sheep [15 ]. Polymerase chain reaction (PCR) amplifications were carried out in a 25-μL reaction volume containing 100 ng of template DNA and 20 pmol of each primer. To reduce the possibility of cross contamination and variation in the amplification reactions, master mixes containing all PCR reagents including the Kapa Taq polymerase enzyme (KAPA Biosystems, Boston, MA, USA) except DNA template were used. The amplification program was performed using the Gene Amp 9700 thermocycler (Applied Biosystems, Warrington, UK). The amplification protocol was an initial denaturation step for 2 min at 94°C, followed by 35 cycles of 94°C denaturation step for 0.5 min, 60°C annealing step for 0.6 min and 72°C extension step for 1 min. The final step was an extension step at 72°C for 5 min. Electrophoresis of the PCR products was done using 1.5% agarose gel and bands were detected by UV lamp after ethidium bromide staining using gel documentation system (Amersham Biosciences, Uppsala, Sweden).

Equal amounts of cell protein lysate (either RIPA or Laemmli lysate; BCA; Pierce) were separated on reducing SDS–PAGE gels, transferred to nitrocellulose or polyvinylidene fluoride membranes, and probed with primary antibody. Bands were visualized and quantified using a Fujifilm Gel Documentation system in combination with HRP-conjugated secondary antibodies and ECL-Plus system (Amersham Pharmacia). Specific activity for Akt and Erk was calculated by normalizing densitrometric values of phosphorylated to total AKT or ERK and E-cadherin. Integrin protein levels were assessed using nonreducing SDS–PAGE gels. VEGF, Il-8, and bFGF levels in the media of 10- to 12-day 3D rBM cultures of MECs were measured using sandwich ELISA (R&D Systems), according to the manufacturer’s instructions. OD measurements were performed using a Fluoroskan Ascent FL (Labsystems).