Nuclear and cytoplasm protein extraction kit

Cardiac fibroblasts/myofibroblasts were lysed with RIPA buffer and proteinase inhibitor mixture (PMSF). Nuclear and cytoplasm protein were separated by using nuclear and cytoplasm protein extraction kit (78833; Thermo Pierce, Rockford, IL, USA), according to manufacturer's instructions. Protein concentration was assessed by Bradford assay (Bio-Rad Laboratories, Shanghai, China). Total proteins were electrophoresed on 12% SDS-PAGE gels and transferred onto polyscreen PVDF transfer membranes (PerkinElmer, Boston, MA, USA). Membranes were blocked with 5% (w/v) non-fat milk in tris-buffered saline (TBS) containing 0.1% Tween 20 for 1 hr at room temperature and incubated overnight with primary anti-col3A1, anti-col1A1, anti-β-actin (sc-28888, sc-8784, sc-47778, respectively, Santa Cruz biotechnology Inc, Santa Cruz, CA, USA), anti-OPN (ab8448; Abcam) at 4°C, respectively. After washing, HRP-conjugated secondary antibody was added for 1 hr at 37°C. Detection was performed with enhanced chemiluminescence (ECL) and relevant blots were quantified by densitometry by using the accompanying computerized image analysis program.

Primary cells were lysed with RIPA buffer and a proteinase inhibitor mixture (PMSF). Nuclear and cytoplasmic proteins were separated using a nuclear and cytoplasm protein extraction kit (78,833, Thermo Pierce, Rockford, IL, USA), according to the manufacturer’s instructions. The protein concentration was assessed using a Multiskan instrument (Thermo, Multiskan Mk3). Total proteins were electrophoresed on 12% SDS-PAGE gels and transferred onto polyscreen PVDF transfer membranes (Immobilon-P PVDF Membrane, Merck Millipore). Membranes were blocked with 5% (w/v) nonfat milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 for 1 h at room temperature and incubated overnight with primary anti-α-SMA (ab32575, Abcam) (1:5000), anti-vimentin (ab92547, Abcam) (1:5000), anti-TLR2 (ab16894, Abcam) (1:1000) and anti-TLR4 (ab22048, Abcam) (1:1000) at 4 °C. After washing, an HRP-conjugated secondary antibody was added for 1 h at 37 °C. Detection was performed with enhanced chemiluminescence (ECL), and blots were quantified by densitometry using the accompanying computerized image analysis program (Image Tool).