P taz

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 CCl₄ and TGF-β1 were purchased from Sigma (St Louis, MO, USA). Antibodies against type I collagen (Affinity, NO. AF7001, 1:1000), α-SMA (Abcam, NO. ab32575, 1:2000), E-cadherin (Sigma, NO. MABT26, 1:1000), BMP-7 (Sigma, NO. MAB4350, 1:1000), Desmin (Proteintech, NO. 16520-1-AP, 1:50000), Vimentin (Proteintech, NO. 60330-1-Ig, 1: 50000), YAP (Proteintech, NO. 13584-1-AP, 1: 5000), TAZ (Proteintech, NO. 23306-1-AP, 1:2000), p-YAP (Affinity, NO. AF3328, 1:1000), p-TAZ (Affinity, NO. AF4315, 1:1000), MST1 (Affinity, NO. DF8430, 1:2000), p-MST1 (Affinity, NO. AF3688, 1:1000), LATS1 (Affinity, NO. AF7669, 1:1000), p-LATS1 (Affinity, NO. AF7169, 1:1000), NF2 (Abcam, NO. ab109244, 1:100000) and GAPDH (Abcam, NO. ab9485, 1:2500) were purchased. Adenoviral vectors expressing the scrambled shRNA (Ad-shCtrl), Ad-shSNHG5, adenoviral vectors expressing a control scrambled sequence (Ad-Ctrl), Ad-SNHG5 and adenoviral vectors expressing NF2 (Ad-NF2) were purchased from Genechem Co.Ltd (Shanghai, China). Animal experimental protocols were approved by the University Animal Care and Use Committee after mice were supplied by the Experimental Animal Center of Wenzhou Medical University.