Amersham ecltm prime detection reagent

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 protein 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^TM-C extra nitrocellulose membranes (Amersham Biosciences), which were blocked of non-specific binding with 5% non-fat milk powder/PBST. Membranes were then probed with rabbit anti-human complex IV (Thermo Fisher Scientific, A-6404, 1:750) or rabbit anti-human lamin B (Abcam, ab16048, 1:500) antibodies before applying HRP-conjugated anti-rabbit IgG antibodies (Jackson ImmunoResearch, 1:2000). Target proteins were visualised using Amersham^TM ECL^TM Prime detection reagent on an Image Quant LAS3000 (GE Healthcare) and images were annotated using Adobe® Photoshop® Elements 5.0.

Cells were treated with inhibitors (5 µM erlotinib or 5 µM cabozantinib) or vehicle control (DMSO) for 24 h prior to harvesting of cell lysates for immunoblotting. This time point was chosen based on previously reported time-course studies in our laboratory [55 (link)]. The protein concentration of cell lysates was quantified by a Lowry assay, and 30 μg/sample was boiled for 10 min with sodium dodecyl sulfate (SDS), subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) at 150 V for 1 h, and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Sigma-Aldrich). Membranes were blocked using 5% bovine serum albumin (BSA) in Tris-buffered saline (TBST) + 0.1% Tween-20. A summary of the primary antibodies used can be found in Supplementary Table S1. Secondary antibodies included goat anti-mouse IgG, or goat anti-rabbit IgG secondary antibodies (Calbiochem, Billerica, MA, USA) conjugated to horseradish peroxidase were used at a concentration of 1:1000, diluted in 5% BSA in TBST. Protein expression was visualized using Amersham^TM ECLTM Prime Detection Reagent (GE Healthcare Lifesciences, Wauwatosa, WI, USA).