Peroxidase conjugated affinipure goat anti rabbit igg

The western blotting analysis was performed as previously described [30 (link)]. In brief, cells were lysed by RIPA buffer containing protease inhibitors and phosphatase inhibitors (Pierce, USA). The protein concentration was measured by a BCA regent kit (Pierce, USA). The lysates were loaded into 10% of sodium dodecyl sulfate-polyacrylamide electrophoresis gels and then transferred to nitrocellulose (NC) membranes (PALL, USA). The membrane was blocked with 5% of bovine serum albumin (BSA) in Tris-buffered saline-Tween 20 and then incubated with a primary antibody at 4°C for overnight. The anti-Flag (F3165) was purchased from Sigma. The β-catenin (sc-7199), TGIF (sc-9084), p21 (sc-397), STIM1 (sc-68897), p53 (sc-6243), Akt (sc-8312), p-Akt (sc-33437), cyclin A (sc-751), cyclin B1 (sc-752), cyclin D1 (sc-718), CDK4 (sc-260), and β-actin (sc-8432) were purchased from Santa Cruz Biotechnology. The Rb (#9313S), phospho-Rb (#8516S), ERK1/2 (#4695S), p-ERK1/2 (#4370S), and PTEN (#9188S) were purchased from Cell Signaling Technology. The secondary antibodies (Peroxidase-Conjugated AffiniPure Goat Anti-Rabbit-IgG and Goat Anti-Mouse-IgG) were purchased from ZSGB-BIO (Beijing, China). The signals were detected in the ChemiDoc™ XRS+ Imaging System (Bio-Rad, USA) by a Bio-Rad Clarity™ western ECL substrate (Bio-Rad, USA).

We washed the cells using ice-cold PBS and lysed in cell lysis buffer (DingGuo, BeiJing) to extract whole-cell protein. The supernatants of cell lysates were collected after cleared by centrifugation (12,000×g for 15 min, 4 ℃). We determined the protein concentrations by BCA protein assay (DingGuo, BeiJing). 15% SDS-PAGE was used to analyzed the protein samples with 40μg, and the samples were transferred electrophoretically to PVDF film (Millipore). Specific antibodies were probed with the blots and the immunoreactive proteins were detected by an ECL kit (Millipore). The antibodies used to carry out the western blot were as followed: Rabbit anti-MCM3 antibody (Proteintech), Rabbit anti-MCM4 antibody (Proteintech), Rabbit anti-E2F1 antibody (Proteintech), Rabbit anti-β-Acting antibody (CWBIO), Peroxidase-conjugated affinipure goat anti-rabbit IgG (ZSGB-BIO). β-Acting was used as an endogenous control and experiments were repeated three times.

Atractylenolide I, (4aS,8aS)-3,8a-dimethyl-5-methylidene-4a,6,7,8-tetrahydro-4H-benzo[f][1] benzofuran-2-one (CAS No.73069-13-3, MF C₁₅H₁₈O₂, m.w. (g/mol) 230.3022, HPLC 98 %) was purchased from Chengdu Best-Reagent Co. Ltd. (Chengdu, China); lipopolysaccharide (LPS), phytohemagglutinin (PHA), MTT (3-[4,5-dimethylthiazol-2-yl]- 2,5-diphenyl tetrazolium bromide), l-kynurenine and 4-dimethylaminobenzaldehyde were purchased from Sigma Chemical Co. (St. Louis, MO, USA); the rabbit polyclonal antibodies to NF-κB p65, phospho-NF-κB p65 (S536)(ab76302), Akt and phospho-Akt (S129) (ab133458), anti-GAPDH antibody [EPR6256] (ab128915) and anti-TLR4/MD-2 complex antibody[7E3](FITC)(ab105855) were purchased from Abcam plc.(Cambridge, MA, USA); Peroxidase-Conjugated AffiniPure Goat Anti-Rabbit IgG was purchased from ZSGB-BIO, Inc. (Beijing,China); ELISA kits for IL-6, IL-4, IL-10, IL-17A, TGF-β1 and VEGF were purchased from R&D Systems, Inc. (Minneapolis, MN 55413, USA); CD4-FITC/CD8-PE/CD3-PerCP (Catalog Number: 340298) for determining CD4/CD8/CD3 counts of T lymphocytes and FoxP3 Staining Kit—FoxP3-PE/CD4-FITC/CD25-APC (Catalog Number: 560133) for determining counts of Treg cells were purchased from Becton, Dickinson and Company (BD Biosciences, USA); CytoTox 96^® Non-Radioactive Cytotoxicity Assay (G1781) was purchased from Promega Corporation (Madison, WI, USA).