Luminata forte western hrp substrate

The BCA technique was used to determine protein concentration. Using SDS-PAGE, proteins were separated and electroblotted upon a PVDF membrane. Protein Free Rapid Blocking Buffer was used to block the transferred membrane. Antibodies were treated with membranes overnight at 4°C. Sixty min were spent incubating the transplanted membrane with goat anti-mouse IgG (H + L) HRP conjugate or goat anti-rabbit IgG (H + L) HRP conjugate. Protein bands were detected using Luminata Forte Western HRP substrate and quantified using a Bio-Rad chemiluminescence imaging system (Bio-Rad, Hercules, CA USA). ImageJ was utilized to quantify the band’s intensity (Rawak Software, Inc., Stuttgart, Germany).

Proteins were isolated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a PVDF membrane. Protein Free Rapid Blocking Buffer was utilized to inhibit the membrane transfer. Antibodies were incubated with membranes overnight at 4 °C. The transplanted membrane was incubated for 60 m with goat anti-mouse IgG (H+L) HRP conjugate or goat anti-rabbit IgG (H+L) HRP conjugate. Using a Bio-Rad chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA), protein bands were detected with Luminata Forte Western HRP substrate and quantified with a Bio-Rad chemiluminescence imaging system (Bio-Rad, Hercules, CA, USA). ImageJ 1.53 was used to measure the intensity of the band (Rawak Software, Inc., Stuttgart, Germany).

Pro-IL-1β and processed IL-1β forms were detected by immunoblotting, as previously described [17 (link), 50 (link), 54 (link)]. Briefly, supernatants were precipitated with 10% trichloroacetic acid (Sigma-Aldrich) and washed in cold acetone by centrifugation at 12,000 × g for 10 min at 4°C. Next, pellets were dried and resuspended in sample buffer. For analysis of cell lysates, the plates were incubated on ice for 10 min, and then the cells were detached with a scraper and transferred into 1.5-ml Eppendorf tubes. Cells were lysed with 50 μl of lysis buffer (50 mM Tris-HCl [pH 7.4], 100 mM NaCl, 1% Triton X-100, and 5% glycerol, all purchased from Sigma-Aldrich) supplemented with a mixture of protease inhibitors (Roche). The lysates were centrifuged at 12,000 × g for 10 min at 4°C, to eliminate cellular debris and proteins were separated on 15% polyacrylamide gels and transferred to nitrocellulose membranes (Bio-Rad). Immunoblotting was performed using goat anti-IL-1β (1:800; R&D Systems). Immunoblots were developed using HRP-conjugated secondary antibodies (1:15,000 R&D Systems) and developed with Luminata Forte Western HRP substrate (Biorad).