To prepare the cell lysates, cells were extracted in RIPA buffer (PH 8) followed by sonication to disrupt the cellular membranes and centrifugation (10,000× g, 7 min at 4 °C) to remove the cellular debris. A total amount of protein of 2 µg was used to perform the analysis.
Cellular supernatants (1 mL) were centrifuged (1200× g, 10 min at 4 °C) to remove the remaining cells, concentrated an average of 4–5 fold times in Amicon Ultra-4 kgl filter units (Merck Milipore, Burlington, MA, USA) and depleted for albumin/IgGs with the ProteoExtract Albumin/IgG removal kit (Merck Millipore, Burlington, MA, USA).
Sample extracts from the cellular/supernatant fraction were resolved by SDS-PAGE and electro-transferred to nitrocellulose membranes. Detection of the protein of interest was performed with antibodies against total IL-1β (monoclonal anti-IL-1β, Cell Signaling, 1:1000) and cleaved IL-1β (monoclonal anti-IL-1β, Cell Signaling, 1:1000) and normalized with β-actin (monoclonal anti-β-actin, Abcam, dilution:1/5000) or total protein (ponceau). Western blot bands were detected by chemiluminescence using a peroxidase enzymatic reaction (Supersignal, Pierce) and quantified with a ChemiDocTM XRS system using the Quantity-One 1-D analysis software (Bio-Rad).
Cellular supernatants (1 mL) were centrifuged (1200× g, 10 min at 4 °C) to remove the remaining cells, concentrated an average of 4–5 fold times in Amicon Ultra-4 kgl filter units (Merck Milipore, Burlington, MA, USA) and depleted for albumin/IgGs with the ProteoExtract Albumin/IgG removal kit (Merck Millipore, Burlington, MA, USA).
Sample extracts from the cellular/supernatant fraction were resolved by SDS-PAGE and electro-transferred to nitrocellulose membranes. Detection of the protein of interest was performed with antibodies against total IL-1β (monoclonal anti-IL-1β, Cell Signaling, 1:1000) and cleaved IL-1β (monoclonal anti-IL-1β, Cell Signaling, 1:1000) and normalized with β-actin (monoclonal anti-β-actin, Abcam, dilution:1/5000) or total protein (ponceau). Western blot bands were detected by chemiluminescence using a peroxidase enzymatic reaction (Supersignal, Pierce) and quantified with a ChemiDocTM XRS system using the Quantity-One 1-D analysis software (Bio-Rad).