Isopropyl 1 thio β d galactoside

Human ASM cells were purchased from ScienCell (Corte Del Cedro Carlsbad, CA, USA). E. coli BL21(DE3) and the pET28(a)+ vector were purchased from Novagen (Germany). The recombinant human CTGF/CCN2 C-terminal domain, Cyr61/CCN1 and NOV/CCN3 were purchased from PROSPEC (USA). The monoclonal mouse anti-human CTGF/CCN2 C-terminus, Cyr61/CCN1 and NOV/CCN3 monoclonal antibody were purchased from R&D system (USA). The anti-6xHis monoclonal antibody was from abcam (UK). The Cell_Light EdU DNA Cell Proliferation Kit was from Ribobio (China). LY294002 (PI3K inhibitor), rabbit antibodies against Akt, mTOR, β-actin, phosphorylated-Akt (Ser473), and phosphorylated-mTOR, and HRP-goat anti-rabbit IgG were purchased from Cell Signaling (USA). NcoI, XhoI, T4 DNA ligase and Easy Taq DNA polymerase were purchased from TaKaRa (Japan). The BCA protein assay kit was provided by Tiangen (China). Cell culture media were purchased from Gibco BRL (USA). The newborn calf serum was obtained from Shiqing (China). Isopropyl-1-thio-β-D-galactoside (IPTG) was purchased from Sigma-Aldrich. The His-bind resin column and total protein extraction kit were purchased from KeyGEN (China).

Antibodies to mouse ZCWPW1 were produced by Dia-an Biological Technology Incorporation (Wuhan, China). Briefly, a complementary DNA (cDNA) fragment encoding amino acids 448 to 622 of mouse Zcwpw1 was inserted into the p-ET-32a + vector (EMD Millipore) and transfected into BL21-CodonPlus (DE3) Escherichia coli cells. The cells were cultured at 37°C overnight and induced by addition of 0.2 mM isopropyl-1-thio-β-d-galactoside (Sigma-Aldrich) for 4 hr at 28°C. Cells were harvested by centrifugation and disrupted by sonication, and the soluble homogenates were purified by Ni-nitrilotriacetic acid (NI-NTA) Agarose (Qiagen) according to the manufacturer’s instructions. The protein was dialyzed in phosphate-buffered saline (PBS) and used to immunize rats, and the antiserum was affinity-purified on antigen-coupled CNBr-activated agarose (GE Healthcare).