Srx 101a developer

Cells were lysed in RIPA buffer (50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholote, 150 mM NaCl, 1 mM EDTA, 1 mM NaF, and proteinase inhibitor cocktail). A total of 200 μg of cell lysate and 10 μl rabbit monoclonal anti-VEGFR-2 antibody (no.2749, Cell Signaling Technology; 1:100 dilution) were incubated in a shaker at 4 °C overnight. After 2 h of incubation with 25 μl of protein A/G ultraLink Resin (Thermo Scientific) at 4 °C, the beads were washed three times with PBS then boiled in SDS-PAGE sample buffer for 5 min to elute proteins for subsequent Western blotting for VEGF R2. For Western blotting, the proteins from placental tissue or first trimester HTR8 trophoblast cell lysates were separated on 12% SDS–polyacrylamide gels and blotted onto PVDF membranes and probed with anti-mouse antibodies for VEGF A, VEGF C, Dll4 (Abcam), Hey 2 and β-actin (Biovision). We used ECL chemiluminescence (Amersham Biosciences) to visualize the bands and recorded them using Konica SRX 101 A developer.

Cancer cells were seeded and cultured until the cells reached 90% confluency. Ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors was used to harvest cell lysates. BCA assay (Thermo Fisher) was performed to determine protein concentration. Proteins were separated using SDS-PAGE and transferred to PVDF membrane (Millipore). Primary antibody incubation was performed at 4 °C for an overnight and secondary antibody was performed at room temperature for an hour. Protein bands were detected using chemiluminescent substrates (Thermo Fisher) on autoradiography films with SRX-101A developer (Konica Minolta).

The NuPage Novex gel system was used for electrophoresis as described in the manufacturer’s protocol (Life Technology, Paisley, UK). Protein samples were resolved on 4%–12% Bis-Tris gels to identify a band size of 66 kD. Proteins were then transferred to a polyvinylidene fluoride (PDVF) membrane purchased from Millipore U.K. Ltd. (Watford, UK), blocked in 5% (w/v) dry milk powder in standard Tris-buffer saline supplemented with 0.1% (v/v) Tween [as recommended by Cell Signalling Technology Inc. (Danvers, MA, USA)] for 4 h. Membranes were incubated at 4°C overnight in primary antibody (1:200 dilutions in 2% (w/v) BSA in TBS-T) then washed twice for 4 min in TBS-T and incubated in the appropriate HRP-conjugated secondary antibody (1:10,000 dilution in 5% (w/v) Marvel in TBS-T) for a minimum of 30 min at room temperature. Membranes were washed four times for 3 min with TBS-T and then incubated in enhanced chemiluminescence (ECL) solution (GE Healthcare Life Sciences, Buckinghamshire, UK) for 1 min. Konica Medical Film was used to visualise the signal, and the exposed films were developed using a Konica Minolta SRX-101A developer.