Nuclear and cytoplasmic protein extraction kit

Nuclear and cytoplasmic proteins were isolated using the Nuclear and Cytoplasmic Protein Extraction Kit (Absin, Shanghai, China). Briefly, ESCC cells were lysed by Cell Fractionation Buffer for 20 min on ice. Then, the supernatants were collected as the cytoplasmic fractions after centrifugation at 4 °C and 15,000×g for 10 min. And the pellets were lysed by Nuclear Isolation Buffer for 5 min on ice. Finally, the supernatants were collected as the nuclear fractions after centrifugation at 4 °C and 15000×g for 10 min. The extracted protein samples were subsequently separated on SDS-PAGE gels and analyzed by WB assays. GAPDH was used as the endogenous cytoplasmic control and Histone H3 was used as the endogenous nuclear control.

We extracted cytoplasmic proteins and nuclear proteins from cardiac tissue samples according to the instructions of the Nuclear and Cytoplasmic Protein Extraction Kit (Absin, Shanghai, China) and quantified for protein levels using a BCA assay kit (Thermo Fisher, Shanghai, China). The specific process has been previously described [16 (link)]. A total of 50 μg protein from heart homogenates was applied per lane for separation by SDS-PAGE. Separated protein was immunoblotted and probed with the following primary antibodies overnight at 4°C: NF-E2-related factor 2 (Nrf2; 1:1000), heme oxygenase-1 (HO-1; 1:1000), Lamin B (1:1000), MR-1 (1:1000), and β-actin (1:1000), all from Abcam, Cambridge, MA, USA. HRP-conjugated secondary antibodies (1:3000, Abcam) were used to label the proteins. Subsequently, we then visualized the bands of target protein using an ECL detection system (Bio-Rad). The information of proteins was normalized to β-actin or Lamin B signals.

Total proteins were extracted from CRC cell lines by using RIPA lysis buffer (Beyotime, China) supplemented with PMSF (Beyotime, China) and phosphatase inhibitor (Roche, Switzerland) and then quantified using BCA Protein Assay Kit (Beyotime, China). Nuclear and cytoplasmic protein were prepared using the Nuclear and Cytoplasmic Protein Extraction Kit (Absin, China) according to the protocol provided by the manufacturer. Protein was electrophoresed through 10% SDS polyacrylamide gels and then transferred to PVDF membranes. The membranes were blocked with 5% fat-free milk for 2 h and incubated with primary antibodies at 4 °C overnight. Membranes were then incubated with second antibodies labeled with HRP at room temperature for 2 h on the following day and the signal was detected using an ECL kit (Beyotime, China). The following primary antibodies were used: JNK (Abcam # ab179461), P-JNK (Abcam # ab124956), c-Jun (CST# 9165), P-c-Jun (Abcam#ab32385), and MMP7 (Abcam#ab205525). β-actin (Abclonal#AC026), GAPDH (Abclonal#AC002) or PCNA (Abcam#ab29) was used as reference gene.