Polytron homogenizer

We disrupted separated lung tissues that had been immediately frozen in liquid nitrogen using a Polytron homogenizer (Pellet pestles cordless motor; Sigma) and centrifuged them. We purified the proteins from the supernatant and assessed the concentrations using the Bradford method. We separated the protein samples by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred them to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech, Little Chalfont, UK). After blocking with 5% skimmed milk (Difco, BD, San Jose, CA, USA), we incubated the membrane overnight with an anti-HDAC2 antibody (1:1,000, rabbit monoclonal; Abcam, Cambridge, UK). We washed the membrane three times with TBST and incubated it with a secondary antibody for 2 hours. Horseradish peroxidase-conjugated goat anti rabbit IgG (1:2,000, Santa Cruz Biotech, Dallas, TX, USA) was used as the secondary antibody. We detected the target proteins using the ECL Western Blotting Analysis System (Thermo Fisher Scientific, Rockford, IL, USA). We either exposed the membrane to X-ray film or analyzed it with an LAS 3000 (Fujifilm, Tokyo, Japan) image analyzer using Multi Gauge v. 3.0 software. We also quantified the HDAC2 and ß-actin band intensities with Multi Gauge.