Imagequant las 3000

Total protein from placental explants and extracellular vesicles were resolved on 14% polyacrylamide SDS-PAGE gels under reducing or non-reducing conditions. Protein lysates were transferred to Hybond^TM-C extra nitrocellulose membranes (Amersham Biosciences, UK). Successful protein transfer was confirmed by staining with 0.1% Ponceau S (w/v). Membranes were blocked with 5% non-fat milk powder (w/v) before incubating with a rabbit polyclonal antibody against human transthyretin (1:500, DAKO, US), rabbit serum IgG as a control, or a mouse monoclonal antibody against β-actin (1:4000, Abcam, NZ). Membranes were then incubated with the corresponding HRP-conjugated anti-rabbit or anti-mouse antibody (Jackson ImmunoResearch, USA) and the presence of target proteins were detected using Amersham^TM ECL^TM Prime detection reagent and visualised on Image Quant LAS3000 (GE Healthcare, UK). Images were annotated using Adobe® Photoshop® Elements 5.0. Protein abundance was semi-quantified by densitometry relative to β-actin using the Kodac Digital Science 1D image analyser (Kodac, Japan).

Total proteins from placental EVs were extracted using RIPA buffer (50 mM Tris, 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Nonidet P40 substitute, 1 mM PMSF, pH7.4) supplemented with protease inhibitor (Roche) and resolved by SDS-PAGE. Proteins were transferred onto Hybond™-C extra nitrocellulose membranes (Amersham Biosciences) which were blocked for non-specific binding by incubating in 5% non-fat milk powder/PBST for an hour at 20°C. Membranes were then probed with rabbit anti-human Flt-1 (Abcam, 1:1,000) or mouse anti-human β-actin (Abcam, 1:5,000) antibodies before applying HRP-conjugated anti-mouse/rabbit IgG antibodies (Jackson ImmunoResearch, 1:2,000). Target proteins were visualized using Amersham™ ECL™ Prime detection reagent on an Image Quant LAS3000 (GE Healthcare). Images were annotated using Adobe^® Photoshop^® Elements 5.0.

Kidney tissues were homogenized using radio-immunoprecipitation assay buffer (Wako Pure Chemicals, Osaka, Japan). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were performed as described previously [24 (link)]. The primary antibodies against 5α-Red1 (Abcam, Cambridge, UK), 11β-HSD2 (Proteintech, Rosemont, USA), and β-actin (Abcam, located in Cambridge, UK) were used. The immunoreactive bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA). The intensities of the specific chemiluminescent bands were analyzed using an ImageQuant LAS 3000 (GE Healthcare, Amersham, UK).