Ecl chemiluminescence detection kit

The treated cells were harvested and lysed with 20% SDS containing 1 mM phenylmethylsulfonyl fluoride. The lysate was sonicated for 1 min on ice followed by centrifugation at 12,000g for 30 min at 4 ºC. Mitochondrial and cytosolic fractions were isolated by using the ProteoExtract^® Cytosol/Mitochondria Fractionation Kit (Merck Millipore, Billerica, MA, USA). Then a sample of protein from the supernatant was resolved by SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking with TBS buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 5% nonfat milk, the membrane was incubated with antibodies against type I procollagen, MMP-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by horseradish peroxidase-conjugated secondary antibodies and then was visualized with an ECL chemiluminescence detection kit (PerkinElmer Life Sciences, Waltham, MA, USA). The relative density of the immunoreactive bands was quantified by using a luminescent image analyzer (LSA-100, Fujifilm, Japan).

DC were washed with calcium- and magnesium-free PBS and lysed in 1 mL of lysis buffer containing 50 mM Tris/HCl (pH 7.5), 150 mM NaCl, 0.5% Nonidet P40, 50 mM NaF, 1 mM sodium orthovanadate, dithiothreitol, and a cocktail of protease inhibitors (cOmplete EDTA-free, Roche). Whole cell lysates were prepared and submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 5%  β-mercaptoethanol using 12% polyacrylamide gels. Harvest of DC and preparation of whole cell lysates were performed as previously described [27 (link)]. Proteins were transferred to polyvinylidene difluoride membranes and immunolabeled using primary antibodies against IκBα, phospho-IκBα, and β-actin. The bound primary antibodies were detected using secondary anti-mouse or anti-rabbit antibodies (GE Healthcare, Little Chalfont, Buckinghamshire, UK) and ECL chemiluminescence detection kit (PerkinElmer, Waltham, MA, USA). The protein bands were scanned and digitized, and the density of each band was determined using the Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).

Mice were sacrificed by cervical dislocation; brains were dissected in ice cold PBS and snap frozen in liquid nitrogen. Prefrontal cortices were homogenized in ice cold HEPES buffer (320 mM Sucrose; 4 mM HEPES, pH 7.3; EDTA with complete protease inhibitors from Roche); cleared by centrifugation at 800×g for 10 min; equal amount of protein was loaded on NuPAGE 4–12% Bis-Tris gel (Novex, NP0322) and blotted detected with HRP conjugated secondary antibodies using a ECL chemiluminescence detection kit (NEL105; PerkinElmer Life Sciences). All antibodies were diluted in 1% BSA TBST buffer. Antibodies: mouse anti-VGluT1 (MAB5502; 1:2000; Chemicon) mouse anti-Synaptophysin (S5768; 1:5000; Sigma); mouse anti-PSD95 (ADI-VAM-PS002-E; 1:5000; Stressgen); rabbit Anti-Tubulin (ab21058; 1:5000; abcam). The density of bands was quantified by densitometry using ImageJ software.