Immunoblotting

The left lung tissues were homogenized in RIPA lysis buffer (HEART, Xi'an, China) containing protease inhibitors, phosphatase inhibitors as well as phenylmethylsulfonyl fluoride (PMSF), and centrifuged at 12,000 rpm at 4 °C for 20 min. Then the supernatant was collected as total protein. The amount of protein in each extract was measured using BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). Subsequently, equal amounts of protein extracts were separated on SDS-PAGE gel (10%) and transferred to polyvinylidene fluoride (PVDF, Bio-Rad, Richmond, CA, USA) membrane using wet transfer. After washing, the blots were incubated at 4 °C overnight with the primary antibodies against HDAC1 (CST; 1:1000), HDAC2 (CST; 1:1000), HDAC3 (CST; 1:1000), tissue inhibitor of metalloproteinase 1 (TIMP-1, Abcam, Cambridge, UK; 1:1000), TIMP-2 (Abcam; 1:1000) and β-actin (Santa Cruz, Dallas, TEX, USA; 1:500). Then the horseradish peroxidase-conjugated goat anti-rabbit IgG (Sigma-Aldrich; 1:5000) was used as the secondary antibodies. Finally, immunoreactive bands were visualized by Super Signal West Pico Chemiluminescent Substrate (Pierce Biotechnology) and quantified by Quality One software (Bio-Rad).