Bca protein assay kit

All solutions were prepared by apyrogenic deionized water (MilliQ System, Millipore, France). All materials used for real-time PCR were purchased from Qiagen (Germany), except the DNA extraction kit that was GF-1 Nucleic Acid Extraction Kit and was purchased from Vivantis Technologies (Malaysia). The GP63 antibody was provided by the Pasteur Institute of Iran, the anti-GFP-HRP goat polyclonal antibody was obtained from Acris antibodies GmbH (Germany), and the beta actin monoclonal antibody was purchased from ProteinTech Group Inc. (Chicago, IL, USA). The nitrocellulose membrane was supplied from Amersham (UK), and the ECL Western blotting substrate was from Pierce™ (Pierce Biotechnology, Thermo Fisher, Rockford, IL, USA). The bovine serum albumin (BSA), phorbol 12-myristate 13-acetate (PMA), Resazurin powder, M199 and RPMI-1640 cell culture media, HEPES, L-glutamine, adenosine, gentamicin, and hemin were sourced from Sigma (Germany). The fetal bovine serum (FBS) and EV-depleted FBS were obtained from Gibco (Life Technologies, Germany). The bicinchoninic acid (BCA) and ELISA kits were purchased from Pierce™ (BCA Protein Assay Kit, USA) and DuoSet ELISA development system (R&D Systems, USA), respectively.

Hs68 cells were treated with vehicle or 1 µM dexamethasone and total protein was isolated using a specific buffer from the Human Phospho-Kinase Array (R&D Systems; Minneapolis, MN, USA) according to the manufacturer’s protocol. The protein concentration in the lysates was quantified using a bicinchoninic acid (BCA) Protein Assay Kit (R&D Systems) and 43 kinase phosphorylation sites were detected on phosphokinase array membranes. The array was rinsed to remove unbound proteins and incubated with biotinylated detection antibodies. Streptavidin–horseradish peroxidase (HRP, Sigma-Aldrich) and electrochemiluminescence detection reagents (Biomax, Seoul, Republic of Korea) were used, and the signals were recorded at each capture spot corresponding to the amount of phosphorylated protein bound using Light-capture (ATTO, Tokyo, Japan).

As described previously [14 (link),25 (link),26 (link)], the cultured cells were homogenized in protein lysis buffer (Bio-rad, Beijing, China), followed by protein concentration assessment with a BCA protein assay kit (R&D systems, Beijing, China). Western blot was performed with the following primary antibodies: rabbit anti-ERK5 (Cell signaling, San Jose, CA, USA), rabbit anti-pERK5 (Cell signaling), rabbit-anti-Stat3 (Cell Signaling) and rabbit anti-pStat3 (Cell Signaling). The secondary antibody was HRP-conjugated anti-rabbit (DAKO, Beijing, China). Image acquisition and densitometric analysis of the gels were performed with NIH ImageJ software (Bethesda, MA, USA).