Cell lysis buffer for western and ip kit

The total protein from tissues and cultured HPASMCs were isolated using cell lysis buffer for Western and IP kit (Beyotime, China) and the nuclear protein and cytoplasmic protein were extracted using nuclear and cytoplasmic protein extraction kit (Beyotime) according to the manufacturer’s protocols. Protein concentrations were determined using the BCA protein assay kit (Beyotime). Proteins were loaded and separated in SDS-PAGE (8, 10 and 15%) and immunoblotted with antibodies including HSP110 (Affinity, USA), LC3 (Abclonal, China), Beclin1 (Abclonal), ATG5 (Abclonal), ATG7 (Abclonal), p62 (Abclonal), p-YAP (Affinity), YAP (Santa cruz, USA), p-TAZ (Affinity), TAZ (Proteintech, China), TEAD4 (Abclonal), β-actin (Santa cruz) and histone H3 (Abgent, USA) overnight at 4 °C. Signal intensities of protein band were developed using ECL chemiluminescence kit (Beyotime) and the band densities were measured using gel imaging analysis system (LiuYi, China). The relative protein levels were calculated after normalization with β-action and compared to the appropriate control group.

The cells were lysed by cell lysis buffer for western and IP kit (Beyotime, China) following the manual of the manufacturers. Alkaline phosphatase assay kit (Beyotime, China) was used to detect the ALP activity in cell lysates. Samples and standards were added in 96-well plates, respectively. Then, para-nitrophenyl phosphate solution and ALP enzyme solution was added in sample and standard wells respectively and incubated for 10 min at 37 °C. Stop solution utilized to terminate the reaction. The 96-well plates analyzed with a microplate spectrophotometer (SpectraMax M2e, Molecular Devices, USA) at 405 nm.
The calcium in cells was detected by calcium colorimetric assay kit (Beyotime, China). The cells were lysed by sample lysis solution in the kit. Following centrifugation at 4 °C (12,000×g, 5 min), the supernatant was used for detecting. Fifty microliters standard and sample were added in 96-wells plates following the manual of manufactures. Test solution (test buffer to o-cresolphthalein complexone = 1:1) was added to each well (150 μl per well) and incubated for 10 min at room temperature in the dark. The 96-well plates analyzed with a microplate spectrophotometer (SpectraMax M2e, Molecular Devices, USA) at 575 nm.

Cell lysis buffer for Western and IP kit and Nuclear and Cytoplasmic Protein Extraction Kit were purchased from Beyotime Biotechnology. Protein lysates were mixed with 5 × SDS sample buffer (Beyotime) and heated at 95 °C for 8 min. Next, a total of 30 μg of protein lysates were run on 12% SDS-PAGE. Proteins were then transferred to PVDF membrane. The membrane was blocked with 5% milk in TBST for 1–2 h at RT and incubated with the primary antibodies overnight at 4 °C. The membrane was next incubated with goat anti-rabbit HRP-conjugated secondary antibody for 1 h at RT. Finally, protein bands were performed with ECL Prime reagent and chemiluminescence signals were detected by Odyssey XF (LI-COR Biosciences). Antibodies used in this study were anti-ZNF143 (Proteintech, 16,618–1-AP, 1:1000), anti-β-actin (Cell Signaling Technology, #4967S, 1:2000), anti-Lamin A/C(ThermoFisher, MA1-06,102, 1:1000), anti-GAPDH (Cell Signaling Technology, #8884S, 1:2000).