Enhance chemiluminescence system

The total protein content was extracted from pulp tissue and HDPFs by using lysis buffer containing protease inhibitors (Sigma-Aldrich, USA). The protein concentration was measured by using a BCA-200 protein assay kit (Pierce, Rockford, Ill., USA). Equal amounts of protein were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was blocked in TRIS-buffered saline with Tween (TBST) containing 5 % nonfat dry milk for 2 h. For the detection of caspase-1, the membranes were probed overnight at 4 °C with a rabbit anti-human caspase-1 antibody diluted 1:100 (Abcam, US) and then incubated for 2 h with a horseradish-peroxidase-conjugated anti-rabbit IgG antibody diluted 1:10,000 (Santa Cruz Biotechnology, Calif., USA). For the detection of NLRP3, the membranes were incubated overnight at 4 °C with a rabbit anti-human NLRP3 antibody diluted 1:100 (Abcam, USA) and then incubated for 2 h with a horseradish-peroxidase-conjugated anti-rabbit IgG antibody diluted 1:10,000 (Santa Cruz Biotechnology). Protein bands were visualized on X-ray film by using an enhance chemiluminescence system (GE Healthcare, Buckinghamshire, UK). The relative protein expression intensities were quantified by densitometry by using Quantity One analysis software.

The total protein content was extracted from the cells by using lysis buffer containing protease inhibitors (Sigma-Aldrich, USA). The protein concentration was measured by using a BCA-200 protein assay kit (Pierce, Rockford Ill., USA). Equal amounts of protein were separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (PVDF). The membrane was blocked in TRIS-buffered saline with Tween (TBST) containing 5% nonfat dry milk for 2 h and probed with primary antibodies myosin (1 : 500, Sigma), α-SMA (1 : 500, Sigma), desmin (1 : 500, Sigma), and GAPDH (1 : 1000, Sigma) overnight at 4°C and then incubated for 2 h with a horseradish-peroxidase-conjugated anti-mouse IgG antibody or anti-rabbit IgG diluted 1 : 20,00 (Sigma). Protein bands were visualized on X-ray film by using an enhance chemiluminescence system (GE Healthcare, Buckinghamshire, UK). The relative protein expression intensities were quantified by densitometry by using Quantity One analysis software.