Mini protean tetra electrophoresis system

At each timepoint collagen extraction from the cell layers and SDS-PAGE was performed [64] (link). Briefly, at days 4, 7, 10 and 13 cell layers were digested with porcine gastric mucosa pepsin (Sigma Aldrich, Ireland) for 2 h at 37 °C with continuous shaking and subsequent neutralisation with 1 N NaOH. The samples were appropriately diluted with distilled water and 5 x sample buffer and heated at 95 °C for 5 min. 10 μl per sample solution per well was loaded on the gel (5% running gel/3% stacking gel). 0.25 mg/ml bovine skin collagen type I (Symatese Biomateriaux, France) was used as reference standard. Electrophoresis was performed in a Mini-PROTEAN Tetra Electrophoresis System (Bio-Rad, Ireland) by applying potential difference of 50 mV for the initial ~30 min and then 110 mV for the remaining time (~ 60 min). The gels were stained using the SilverQuest™ (Invitrogen, Ireland) silver stain kit, according to the manufacturer's protocol. Images of the gels were taken after brief washing with water. To quantify the cell-produced collagen type I deposition, the relative densities of collagen α1(I) and α2(I) chains were evaluated using ImageJ software (NIH, USA) and correlated to the α1(I) and α2(I) chain bands densities of the reference standard collagen type I.

Cell extractions were mixed with 2× reducing sample buffer (100 mM Tris [pH 6.8], 10% β-mercaptoethanol, 4% SDS, 0.3% bromophenol blue, 20% glycerol) and boiled in a heat block at 100°C for 5 min. Samples were loaded on polyacrylamide gels using 1× running buffer (25 mM Tris, 20 mM glycine, 0.05% SDS) and run on a Mini-Protean Tetra electrophoresis system (Bio-Rad) with a constant voltage of 100 V for 90 min.

Cobrotoxin was a gift from the Department of Pharmacy at the Suzhou University Medical College (Suzhou, Jiangsu, China), while the 3-methyl adenine (3-MA), dimethyl sulfoxide (DMSO) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). SB203580 was purchased from Beyotime Institute of Biotechnology (Haimen, China) and an automatic enzyme standard instrument (Benchmark) was purchased from Bio-Rad (Hercules, CA, USA). A Heracell 150 carbon dioxide incubation box was purchased from Thermo Fisher Scientific (Rockford, IL, USA). SDS-PAGE apparatus (Mini-PROTEAN Tetra Electrophoresis System and Mini-PROTEAN 3 Dodeca Cell, Bio-Rad) and a FEI TECNAI 10 transmission electron microscope (TEM; Philips, Amsterdam, Holland) were also utilized.

A Native-PAGE was applied to analyze the molecular structures of the untreated and sonicated OVM. The precast BeyoGel™Plus PAGE (Beyotime Biotechnology Co. Ltd, Shanghai, China) included 4 % stacking gels and 15 % separating gels. 80 μL of sample was mixed with 20 μL loading buffer (4 × ). Gel board was mounted into a Biorad Mini-PROTEAN Tetra Electrophoresis System (Biorad, California, US), and 20 μL of each sample mixture was loaded into gel lanes. The running buffer was prepared by Tris-Gly Native-PAGE buffer powder. A color mixed protein marker of 11–180 kDa (Solarbio, Beijing, China) was utilized to estimate the molecular mass of each sample. The whole electrophoresis process kept the operating voltage at 150 mV.

To measure the expression levels of the split TEV GPCR fusion constructs, the plasmids were transfected into HEK-293 cells using Lipofectamine 3000 according to the manufacturer’s instructions. After allowing the plasmids to express for 24 h, the cells were washed 1× with PBS and lysed in a Triton-X lysis buffer (1% Triton-X100, 50 mM Tris pH 7.5, 150 mM NaCl, and 1 mM EGTA) containing the Complete protease inhibitor cocktail (Roche, Basel, Switzerland) and the PhosSTOP phosphatase inhibitor (Roche). The lysed cells were kept on ice for 10 min, sonicated 3× for 10 s at 4 °C, and denatured for 10 min at 70 °C. The Mini-PROTEAN Tetra Electrophoresis System (Bio-Rad, Hercules, CA, USA) was used for running and blotting the protein gels. For the chemiluminescence detection of proteins, the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) was used followed by imaging with a ChemoStar ECL imager (Intas Science Imaging Instruments, Göttingen, Germany). The HA-tagged proteins were visualized using an HA antibody (clone 3F10, dilution 1:500, No. 11 867 423 001, Roche).

After the washing procedure (described in Supplementary Methods), the proteins surrounding the graphene were denatured by boiling for 5 min in blue loading buffer composed of 62.5 mM Tris-HCl (pH 6.8 @ 25uC), 2% (w/v) SDS, 10% glycerol, 0.01% (w/v) bromophenol blue, and 40 mM DTT. After this procedure, the corona proteins were denatured and coated with SDS surfactant (which provides them with negative net charge). The samples were loaded in a 10% polyacrylamide gel (1D SDS-PAGE), and separated by size upon application of an electric field using a Mini-PROTEAN Tetra electrophoresis system from Bio-Rad. A constant voltage of 130 V was applied for about 45 min of the electrophoretic run. In order to visualize the protein bands, all the gels were stained using 2D-SILVER STAIN-II reagents (Cosmobio Co.,Ltd) prior to scanning under white light using a G:Box Chemi XT4 (Syngene). Uncropped scans of the gels are reported in SI.