Sc 74443

Pleiotrophin (PTN) levels in 80 serum samples from four different patient groups (cancer-free, CPG1 and CPG5, and patients with metastatic PC) were determined by Sandwich ELISA. Diluted in 0.2 M sodium bicarbonate (pH 9.4), commercial PTN capture antibody (sc-74443, 1:250, Santa Cruz) was coated in 96-well, half-area plates (#3690, Corning) overnight at 4 °C. Then, the plates were washed three times with phosphate-buffered saline (PBS) and blocked with 5% bovine serum albumin in PBS overnight at 4 °C. Serum samples (4 μL) were incubated at room temperature for 2 h. Plates were then washed three times with PBS containing 0.01% Tween-20 (PBST) and were then incubated with biotinylated PTN detection antibody (BAF252, 1:100, R&D Systems) at room temperature for 2 h. Plates were washed three times with PBST, incubated with peroxidase-conjugated streptavidin biotin (PI21134, 1:200, Thermo Scientific) at room temperature for 1 h, washed three times with PBST and washed another two times with PBS. Bound proteins were detected using ultra TMB-ELISA substrate (#34028, as per the manufacturer’s directions, Thermo Scientific) and analysed by a Promega plate reader at 450 nm. The standard curve for PTN concentrations was determined using 0.1–200 ng/ml recombinant human PTN (252-PL-050, R&D Systems).

After washing cells twice with ice-cold PBS, cells were incubated on ice for 15 min in protein lysis buffer containing 20 mM HEPES, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 2 mM Na₃VO₄, 10 mM NaF, and protease inhibitor cocktail (P8340, Sigma-Aldrich). Cells were harvested using a cell scraper, and cell lysates were prepared by centrifugation at 13,000 rpm for 15 min at 4 °C. Supernatant was collected and protein concentrations were determined by Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Samples were resolved by SDS-PAGE and transferred to PVDF membrane (EMD Millipore). After blocking membranes in 5% skim milk containing TBS-T (w/v) for 1 h at room temperature, membranes were incubated in primary antibodies (PTN, 1:500 dilution in 2% BSA containing TBS-T, sc-74443, Santa Cruz and β-actin, 1:2000 dilution in 2% BSA containing TBS-T, A1978, Sigma-Aldrich) overnight at 4 °C. Membranes were washed three times for 10 min in TBS-T and incubated in anti-mouse HRP-conjugated secondary antibody (1:3000 dilution in TBS-T, Bio-Rad, Hercules, CA, USA) for 1 h at room temperature. Following washing, membranes were developed using SuperSignal West Femto Substrate (Thermo Fisher Scientific) and scanned using ImageQuant LAS 4000 (GE Healthcare, Little Chalfont, UK).