Eclplus

HCE-2 cells were washed with cold 0.01 M PBS, pH 7.4 at the end of the treatment and dissolved in 1% SDS. The BCA (bicinchoninic acid) protein assay was used to quantify the total protein levels. Lysates (20 μg/lane of protein) were resolved by electrophoresis on a 4–20% SDS-polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred onto nitrocellulose membranes. Blots were blocked for 1 h at room temperature in 20 mM Tris-buffered saline, pH 7.6, 0.1% Tween 20 (TBS-T) containing 5% non-fat dry milk, and then incubated overnight at 4 °C with rabbit polyclonal antibodies against IL-1β and COX-2 (all from Abcam, Cambridge CB2 0AX, UK), monoclonal rabbit antibodies against Phospho-NF-κB p65 (Ser536) (93H1) (Cell Signaling Technology, Beverly, MA, USA), diluted 1:1000 in TBS-T containing 5% bovine serum albumin. As loading control, the monoclonal anti-β-actin antibody was from Sigma (St. Louis, MO, USA). Immunodetection was performed with secondary antibodies (1:3000 anti-mouse or antirabbit IgG from donkey, Amersham Biosciences, Amersham, UK) conjugated to horseradish peroxidase in TBS-T containing 5% non-fat dry milk. Membranes were washed with TBS-T and then reactive bands were detected using chemiluminescence (ECLplus; Euroclone, Padova, Italy). Quantitative analysis was performed using the QuantityOne analysis software (Bio-Rad, Hercules, CA, USA).

Western blotting was conducted
as previously reported.^{34 (link)} Four slices of
the sample were dissolved in 1% SDS. BCA (bicinchoninic acid) protein
assay was used to quantify the total protein levels. Lysates (20 μg/lane
of protein) were resolved by electrophoresis on a 4–20% SDS-polyacrylamide
gel (Bio-Rad Laboratories, Hercules, CA, USA) and transferred onto
nitrocellulose membranes. After blocking, the blots were incubated
overnight at 4 °C with rabbit-polyclonal anti-PDE9 (Millipore,
Burlington, Massachusetts, USA) diluted 1:1000 in TBS-T containing
5% nonfat dry milk. β-Tubulin was used as a loading control
(monoclonal antibody) purchased from Sigma (St Louis, MO, USA). Immunodetection
was performed with HRP-conjugated secondary antibodies (1:2000) antimouse
or antirabbit IgG from donkey (Amersham Biosciences, Little Chalfont,
UK) in TBS-T containing 5% nonfat dry milk. After the membranes and
reactive bands were washed, they were detected using chemiluminescence
(ECLplus; Euroclone, Padova, Italy). Quantity One analysis software
was used for quantitative analysis (Bio-Rad, Hercules, CA, USA). Results
are presented as the mean standard error of the mean (SEM) of different
gels and expressed as AU-, which depicts the ratio between the levels
of target protein expression and β-tubulin normalized to basal
levels.