Anti pde4b

The lung tissue was homogenized (1/10 w/v) using a homogenizer in tissue lysis/extraction reagent (Sigma-Aldrich) supplemented with protease inhibitors (Sigma-Aldrich). Immunoblotting was performed as described previously [12 (link)]. The following primary antibodies were used: anti-PDE4B (1:1000 dilution; Abcam), anti-MMP-9 (1:1000 dilution; Abcam), and anti-β-actin (1:1000 dilution; Cell signaling, Denver, MA, USA). Expression of PDE4B, MMP-9, and β-actin were evaluated using ChemiDoc (Bio-Rad, Hercules, CA, USA).

Murine T cells were centrifuged at 400x g for 4 min at 4°C, washed twice with ice-cold PBS, and lysed in RIPA buffer supplemented with Halt protease inhibitor cocktail (100x, Thermo Scientific). An equal number of cells was used continuously throughout the experiment. Samples were boiled at 95°C for 5 min. Protein lysates were loaded on 10% gel and SDS-PAGE was run followed by protein transfer onto nitrocellulose membrane (Amersham). Membranes were blocked in 3% or 5% non-fat milk prepared in TBS-T buffer, depending on the antibody, for 1 h at room temperature (RT). Subsequently, indicated primary antibodies were used in overnight incubation at 4°C: anti-PDE2A (1:750, Fabgennix), anti-PDE3B (1:2000, kindly provided by Dr. Sergei Rybalkin), anti-PDE4B (1:2500, Abcam) and anti-PDE4D (1:2500, Abcam). Anti-GAPDH (1:160000, Bio Trend) was used as a loading control in 30 min incubation at RT. After probing with the primary antibody, membranes were washed with TBS-T buffer three times and incubated for 1 h at RT with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, goat anti-mouse or goat anti-rabbit, Biorad). To detect the signal, the SuperSignal West Pico PLUS Kit (Thermo Scientific) was used according to the manufacturer’s protocol. Densitometry analyses were performed on scanned blots in ImageJ software.