Luminata forte chemiluminescence

IL-6 and Il-1β levels were determined in the supernatant by western blotting. The supernatant was collected after treatment and protein was precipitated using 4 volumes of ice-cold acetone. The protein was dissolved in RIPA buffer containing protease inhibitors before determining the concentration of cellular protein using the Pierce Coomassie Bradford protein assay (Thermo Fisher Scientific, Cat. #23236). Samples were separated on a 4-15% gel before being transferred to a PVDF membrane to probe for IL-1β (1:500), IL-6 (1:500), and β-actin (1:2000) as previously described (27 (link)). Blots were incubated with primary antibodies overnight at 4°C before being treated the following day with anti-rabbit secondary horse radish peroxidase-conjugated antibodies (1:10,000 for 1 hr at room temperature; Abcam, Cambridge, UK). Membranes were washed, incubated with a Luminata Forte Chemiluminescence (Millipore) for protein detection, and analyzed using an ImageQuant LAS 4000 imager and software (GE Healthcare Life Sciences, Marlborough, MA).

Cells were cultured in 100% PM or in 75% PM supplemented with 25% MΦ-CM. After 48 h, the cells were lysed, and the cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose membranes, immunoblotted with anti-cleaved caspase-3 antibody (Cell Signaling Technology) followed by horseradish peroxidase-linked goat anti-rabbit immunoglobulin G (IgG) antibody (Cell Signaling Technology), and revealed with Luminata Forte chemiluminescence (Millipore, Schaffhausen, Switzerland) [29 (link)]. As a loading control, membranes were also immunoblotted with anti-α-tubulin antibody (Sigma-Aldrich) followed by horseradish peroxidase-conjugated sheep anti-mouse IgG antibody (Amersham, Dietikon, Switzerland) and revealed with Luminata Crescendo chemiluminescence (Millipore). Blocking antibodies were from InvivoGen (anti-IL-6) and R&D Systems (anti-tumor necrosis factor α (anti-TNF-α)).