Enhanced chemoluminescence

To characterize H441 cell line as appropriate positive control, a Western blot analysis was performed. Cells were washed twice in cold PBS, collected and, after centrifugation, solubilized in 10 mMTris-HCl, 160 mMNaCl, 1 mM EGTA, 1% deoxycholic acid, 1% Triton, 0.1% SDS and a complete mini-protease inhibitor cocktail tablet (SIGMA Chemicals Company). The cells were left to lyse one ice for 30 minutes and, after centrifugation at 12000 g for 30 minutes, the supernatant was then collected. Protein concentration was determined by bicinchoninic acid (BCA) assay. Protein extract (30 μg) was incubated at 100 °C for 10 minutes and separated on 4–12% SDS-PAGE gel, blotted onto PVDF membrane (GE Healthcare) and probed overnight at 4 °C with following primary antibodies: mouse monoclonal anti-pan-cytokeratins (2A4), mouse monoclonal anti-PD-L1 (1B12), mouse monoclonal anti-IgG1 (isotype control) (NCG01) (Abcam) and mouse monoclonal anti-EpCAM (C-10) (Santa Cruz Biotechnology). Membranes were washed three times for 10 minutes in TBS and 0.1% Tween-20 and left to incubate with the appropriate secondary antibodies for 1 h at room temperature. After three washes, immunoreactive bands were visualized by enhanced chemoluminescence (PerkinElmer). Mouse anti-actin (Santa Cruz Biotechnology) served as a protein loading control.

Total protein extracts were obtained in RIPA buffer (50 mmol l⁻¹ Tris (pH 8.0), 150 mmol l⁻¹ NaCl, 0.5% (w/v) sodium deoxycolate, 0.1% (w/v) SDS, 1% (v/v) NP40, 0.001 mol l⁻¹ EDTA and a mix of protease inhibitors). Protein extracts were separated by SDS-PAGE and blotted onto nitrocellulose membranes (Perkin Elmer). Membranes were blocked with 5% (w/v) nonfat dried milk and incubated with primary antibodies (Abs) at the appropriate dilutions. Abs were as follows: goat anti-β-actin, rabbit anti CCND1 and mouse anti-MYCN (Santa Cruz Biotechnology), mouse anti-Gli1, no. 2643 (Cell Signaling Technology Inc). Immunoreactive bands were visualized by enhanced chemoluminescence (Perkin Elmer).
Densitometric analysis was carried out using ImageJ. Actin normalized optical density results are reported as numbers under the relevant bands and summarized as bar plots in electronic supplementary material, figure S8.

In order to analyze MRP4 protein, cells were washed twice with cold PBS, collected, and centrifuged at 400 g for 10 min. Cell pellets were then resuspended in lysis buffer (RIPA buffer: 10 mM Tris-HCl (pH 7.6), 160 mM NaCl, 1 mM EGTA, 1% deoxycholic acid, 1% Triton, and 0.1% SDS) with protease inhibitors, incubated on ice for 30 min, and centrifuged at 12,000 g for 30 min; the supernatant was then collected.
Protein extracts (30 μg) were incubated at 37°C for 30 min [20 (link)] and separated on 4–12% SDS-PAGE gel, blotted onto PVDF membrane (GE Healthcare, Milano, Italy), and probed with rat anti-MRP4 (Alexis, Plymouth Meeting, Pennsylvania) and mouse anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA) monoclonal antibodies. Immunoreactive bands were visualized by enhanced chemoluminescence (PerkinElmer, Waltham, MA, USA).

Total protein was isolated from primary osteoblast and osteoclast cultures. Protein isolation and Western blotting was performed as described in Verlinden et al. (9 (link)) Briefly, total protein extracts were isolated and 20 µg of protein was separated by SDS-polyacrylamide gel electrophoresis (PAGE) using 4-12% polyacrylamide gels (Thermo Fisher Scientific) and transferred to a nitrocellulose membrane (GE Health care). Membrane blocking was done in TBS (10 mM Tris-HCl, pH 7.6; 150 mM NaCl) with 5% non-fat dry milk and 0.1% Tween 20 (Merck). The rabbit monoclonal (D39A5) anti-mouse NRP2 antibody (CST) was diluted 1/500, while the mouse anti-actin (Merck) was diluted 1/5000. Incubation with a secondary antibody (Dako) was performed for 1 h at RT, and enhanced chemoluminescence (Perkin Elmer) was used to visualize protein bands.