Odissey scanner

Crude synaptosomal fractions were obtained as described (Mlewski et al., 2008 (link)). All samples were valued for protein concentration (DC Protein Assay Kit, Bio-Rad Laboratory, Hercules, CA, USA) and equal protein amounts (25 μg) were separated into 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to Nitrocellulose membranes (Bio-Rad Laboratory, Hercules, CA, USA) as previously described (Krapacher et al., 2010 (link)). Membranes were incubated overnight at 4°C with their primary antibodies diluted in 1% skim milk in TBST. The antibodies used were anti-α-tubulin (1:3000, DM1A; Sigma-Aldrich, St. Louis, MO, USA) and anti-spectrin (1:1000, Millipore Corporation, Billerica, MA, USA). Membranes were then washed three times in TBST and incubated with a Licor secondary antibody (1:15,000, IRDye^® 800CW LI-COR Biosciences, Lincoln, NE, USA) or a horseradish peroxidase–conjugated antibody (1:2000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. After two washes with TBST and two washes with TBS, bands were visualized using Odissey scanner (LI-COR Biosciences, Lincoln, NE, USA) or a chemiluminescence detection kit (ECL; Amersham Life Science, Buckinghamshire, England) according to the primary antibody used.

Proteins from CD2⁺-T cells and CD14⁺ monocytes of MM patients and controls were solubilized with lysis buffer [50 mM Tris(Tris(hydroxymethyl)aminomethane)-HCl (pH 8.0), 150 mM HCl, 5 mM ethylenediaminetetraacetic acid, 1% NP40 and 1 mM phenylmethyl sulfonyl fluoride]. The protein concentration was measured by DC™ Protein Assay (Bio-Rad, California, U.S.). Cell proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and afterwards transferred to nitrocellulose membranes (Millipore, Massachusetts, U.S.). The membranes were incubated overnight at 4°C with mouse anti-LIGHT, mouse anti RANKL (Abcam, Cambridge, U.K.) and rabbit anti-total ERK (Santa Cruz Biotechnology, Texas, U.S.). After incubation with appropriate IRDye 800 cw goat anti-mouse and IRDye 800 cw goat anti-rabbit secondary Ab (LI-COR, Nebraska, U.S.), the membranes were detected on an Odissey scanner (LI-COR, Nebraska, U.S.).