Sc12730

2x10⁴ MM cells were seeded on glass coverslips and the following day treated overnight with either BoxA (400 nM) or CXCL12 (10 nM) and PBS (control). Following treatment, cells were fixed with 4% paraformaldehyde in PHEM buffer for 10 min at RT, washed twice with 1% BSA in PBS for 5 min, and then blocked with 4% BSA and 10% goat serum in PBS. Cells were overlayed with the primary antibodies:
rabbit monoclonal anti‐CD47 (1:100, EPR21794, Abcam #AB218810); mouse monoclonal anti‐CD47 (1:50, B6H12, Santa Cruz #sc12730) either alone or in combination or goat polyclonal anti‐CXCR4 (1:100, Abcam #AB1670), or rabbit monoclonal anti‐TLR4 (1:50, Cell signaling #14358) and or rabbit polyclonal anti‐RAGE (1:100, Invitrogen #PA1‐075) for 1 h at room temperature. Following three washes with 0.2% BSA in PBS, the cells were incubated with secondary antibodies in 0.2% BSA/PBS + 10% goat serum and incubated for 45 min at RT. For nuclei staining, 1 µg/ml Hoechst 33358 was used. For cytosol detection, Phalloidin FITC (P5282, Sigma‐Aldrich) was used.
Secondary probes (Duolink, Sigma‐Aldrich) for PLA reaction were as follows: Anti‐Rabbit MINUS (#DUO92005), Anti‐Rabbit PLUS (#DUO92002), Anti‐Goat MINUS (#DUO92006), and Anti‐Goat PLUS (#DUO92003). When both primary antibodies were used, the PLA products were obtained by using the Anti‐Rabbit PLUS and Anti‐Goat MINUS probes.

Adherent cells were washed twice in cold phosphate-buffered saline and lysed in RIPA buffer (Thermofisher, 89900) (50 mM Tris-HCl pH 8, 150 mM NaCl, 1% Nonidet, P-40, 0.5% sodium deoxycholate and 0.1% sodium dodecyl) with freshly added protease inhibitor mixture (Roche, 5056489001). Cells debris were removed by centrifugation at 9000 g for 20 min at 4 °C. A micro BCA assay was used to quantify protein concentration. Cell lysates were precleared with dynabeads protein G magnetic beads (Thermofisher, 10003D). Immunoprecipitation was performed overnight at 4 °C with 40 µl of dynabeads protein G that were preincubated with 8 µL of a rabbit anti-β3 integrin subunit antibody (Sigma, AB2984, 1 mg/ml). After washing the beads with the lysis buffer, immunoprecipitated proteins were eluted with a SDS sample buffer under non-reducing conditions. They were analyzed by western blotting after separation on 10% SDS-polyacrylamide gel electrophoresis using the mouse monoclonal anti-CD47 antibody (B6H12, Santa Cruz, sc-12730, dilution 1:200) and a horseradish peroxidase-conjugated secondary antibody for chemiluminescence detection (Sigma, A0545, dilution 1:3000).