Ecl chemiluminescence

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.

Cultured MDDCs (1×10⁶) were suspended in a lysis buffer containing 20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1% Triton X-100, and protease inhibitors. The lysates were then subjected to sodium dodecyl sulfate-polyacrylamide (SDS) gel electrophoresis, after which they were transferred to nitrocellulose membranes that were blocked for 1 hour at 25°C with a blocking buffer and then incubated with primary Abs in blocking buffer overnight at 4°C. The samples were treated with secondary Abs in a blocking buffer. Signals were detected by ECL chemiluminescence (Amersham, Uppsala, Sweden).

Purified hPTHrP1-34 and 1-84 and proteins extracted from kidney were quantitated by a kit (Bio-Rad, Mississauga, Ontario, Canada). Thirty µg protein samples were fractionated by SDS-PAGE and transferred to nitrocellulose membranes. Immunoblotting was carried out as described [22] (link) using antibodies against PTHrP1-34, TRPV5 (ECaC1), calbindin-D_9K and calbindin-D_28K, Na⁺/Ca²⁺ exchanger 1 (NCX1), and β-tubulin (Santa Cruz, CA, USA). Bands were visualized using ECL chemiluminescence (Amersham) and quantitated by Scion Image Beta 4.02 (Scion Corporation, NIH).

Proteins were extracted from mandibles and quantitated using a protein assay kit (Bio‐Rad, Mississauga, Ontario, Canada). Protein samples (20 μg) were fractionated by SDS‐PAGE and transferred to nitrocellulose membranes. Immunoblotting was carried out as described¹⁹ using antibodies against Cyclin D (1:300, sc‐8396, Santa Cruz), CDK4 (1:300, sc‐23896, Santa Cruz), p27 (1:500, sc‐1641, Santa Cruz), p53 (1:500, sc‐126, Santa Cruz) and β‐actin (1:1000, AP0714, Bioworld Technology). Bands were visualized using ECL chemiluminescence (Amersham) and quantitated by Scion Image Beta 4.02 (Scion Corporation, NIH).

Equal amounts of total cell lysate proteins were loaded, separated by 10% SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Membranes were incubated with primary antibodies against GDF15 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h and subsequently with horseradish peroxidase-conjugated secondary antibodies for 1 h. Band densities were detected by ECL chemiluminescence (Amersham Biosciences, Buchs, Switzerland) as described by the manufacturer. Tubulin (Cell Signaling Technology, Beverly, MA, USA) was used as an internal control. Images were scanned with a master imager (Microtek ScanMaker 9800XL, Shanghai, China) and semi-quantified with Photoshop 7.0 (Adobe, San Jose, CA, USA).