To functionally express and determine the

To functionally express and determine the ligand selectivity of the stingray (sr) MC1R, srMC2R, srMC3R, srMC4R, and srMC5R paralogs, the nucleotide sequences for the srmcrs were separately synthesized with a V-5 epitope tag at the N-terminal of the receptor, and inserted into a pcDNA3.1 expression vector (GenScript; Picataway, NJ, USA). Each srmcr cDNA was separately transiently transfected into Chinese Hamster Ovary (CHO) cells. The CHO tachykinin receptor were grown at 37°C in a humidified 5% CO2 incubator in DMEM/F12 with 5% fetal calf serum. Each sr cDNA was co-expressed with a CRE/Luciferase reporter plasmid [2] using the Solution T Cell Line Nucleofector Kit (Amaxa Inc., Gaithersburg, MD, USA) and program U-23 [4]. The transiently transfected cells were seeded on a 96-well plate at a density of 1×10−5 cells/well. After 48h in culture, the transfected cells were stimulated with either synthetic srACTH(1-24), srDes-acetyl-α-MSH, srβ-MSH, srγ-MSH, srδ-MSH, srβ-endorphin or hACTH(1-24), or NDP-MSH at concentrations ranging from 10−6M to 10−12M, in serum-free CHO media for four hours at 37°C. At the end of the incubation period, 100µl of Bright-Glo luciferase assay reagent (Promega Inc., Madison, WI, USA) was added to each well, and incubated for 5min at room temperature. Luminescence was measured with a Bio-Tek Synergy HT plate reader (Bio Tek, Winooski, VT, USA), and the dose response curves were analyzed by using Kaleidagraph software (Synergy Software, Reading, PA, USA). All experimental treatments were performed in triplicate.
Acknowledgments We thank Mr. Kosuke Arai, Kitasato University, and Dr. Yasuhisa Kobayashi, Dr. Naoaki Tsutsui and Mr. Kazuhiro Saito, Okayama University, for technical assistance. Partial support for this study was provided by the Long Research Fund (RMD) and National Science Foundation, U.S.A. Grant IOB 0516958 Supplement (RMD).
Specifications table
Value of the data
Data Here, we exemplified the application of laccase in destaining of CBBR-stained polyacrylamide gels by using laccase from Cerrena sp. HYB07 [2], and various parameters were evaluated based on gel background intensity and protein band signals after destaining. Among the laccase mediators tested, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) resulted in low gel background intensity and strong protein signal (Fig. 1). With ABTS as the mediator, effect of temperature, laccase activity, ABTS concentration and destaining time on gel destaining was successively studied (Figs. 2–5). The optimal temperature, laccase activity, ABTS concentration and destaining time were determined to be 37°C, 15UmL−1, 2μM and 2h, respectively.
Experimental design, materials and methods Fermentation of Cerrena sp. HYB07 was conducted in a 5L fermenter for 7d as previously described [1]. Fermentation broth was harvested by pressure filtration and diluted in distilled water for polyacrylamide gel destaining. Laccase activity was assayed with ABTS (Sigma-Aldrich) as the substrate at pH 3.0 and 40°C. One unit of enzyme activity (U) was defined as the amount of laccase required to oxidize 1μmol ABTS in 1min. Bovine serum albumin (BSA) at 500ng was separated by SDS-PAGE by using a Mini-PROTEAN Tetra Cell (BioRad, USA) on 5% stacking gels and 12% separating gels (1mm thick). Staining solution contained 0.05% (w/v) CBBR (Sangon Biotech, Shanghai, China) in distilled water. Unless otherwise stated, the gel was stained by boiling in the staining solution for 1min and destained in 50mL destaining solution with shaking. The destaining solution was not changed during destaining. Gel background intensity and protein band areas were measured with ImageJ [3]. To choose the suitable mediator for laccase-mediated destaining of CBBR-stained polyacrylamide gels, different mediators were added to the destaining solution (distilled water supplemented with 20UmL−1 laccase). Acetosyringone (ACE), syringic acid (SYA), 1-hydroxybenzotriazole (HBT) and syringaldehyde (SYD) were used at the final concentration of 20μM, and ABTS at 2μM at 25°C.