Mini protean precast gel

Whole-cell lysates were prepared in RIPA buffer (Sigma Aldrich) supplemented with phosphatase and protease inhibitors (Roche, Indianapolis, IN, USA). Protein quantification was performed with the DC Protein Assay kit (Bio-Rad, Hercules, CA, USA) and 20 μg of proteins were loaded in 4–15% Mini-PROTEAN precast gels (Bio-Rad). The following primary antibodies were used for western blotting: anti-MYC (Epitomics, Burlingame, CA, USA; rabbit, 1:2000), anti-BRD4 (Epitomics; rabbit, 1 : 1000), BCL2 (Cell Signaling Technology, Danvers, MA, USA; rabbit, 1 : 2000), GAPDH (Cell Signaling Technology; rabbit, 1 : 5000), Cleaved-Caspase3 (Cell Signaling Technology; rabbit, 1 : 2000), LC3B (Cell Signaling Technology; rabbit, 1 : 1000), Bak (Cell Signaling; 1 : 1000). Each western blot data have been confirmed in at least two biological independent experiments.

The renal protein expressions of protein kinase C alpha (PKC-α) and nuclear factor erythroid 2-related factor 2 (NRF2) were determined via Western blot analysis, as described previously [20 (link),22 (link)]. Briefly, protein extracts from the renal cortex were electrophoresed on 4–20% Mini-PROTEAN precast gels (Bio-Rad Laboratories, Richmond, CA, USA) under non-reducing conditions. The blots were incubated with PKC-α (rabbit monoclonal; catalog no. ab32376, Abcam) and NRF2 (goat polyclonal, catalog no. SAB2501713; Sigma-Aldrich-Merck) overnight at 4 °C, followed by incubation with goat anti-rabbit or rabbit anti-goat secondary antibodies, respectively (Dako Corp., Carpinteria, CA, USA). Membranes were subsequently probed for β-actin (Sigma-Aldrich) to confirm equal loading of samples. An ECL detection kit (Sigma-Aldrich) was used for the detection of blots and quantified via densitometry using Quantity-One software 4.6 (Bio-Rad Laboratories).

Cells were washed twice in chilled 1X PBS and lysed in chilled 1X RIPA buffer supplemented with protease (Roche, Cat No. 04 693 116 001) and phosphatase inhibitors (Roche, Cat No. 04 906 837 001). Protein concentrations were determined using Pierce BCA assay kit. Samples were run on 4-20% Mini-PROTEAN pre-cast gels (Biorad, Cat No. 4568094) and transferred to PVDF membranes. The RhoActivity assay was performed using the RhoA pull-down activation assay biochem kit (Bead pull-down format) (Cytoskeleton, Cat No. BK036). The assay was carried out by incubating 800 μg of total cell lysate with 50 μg of Rhotekin as described in the manufacturers protocol following which western blots were performed. Antibodies used and dilutions are as follows: RhoA 1:1000 (Cell Signaling Technology, 2117), β-actin 1:5000 (Cell Signaling Technology, 3700S), Myosin Light Chain 2 1:1000(Cell Signaling Technology, 3672), Thr18/Ser19 Phospho-Myosin Light Chain 2 1:1000(Cell Signaling Technology, 3674), GLUT1 1:1000 (Cell Signaling Technology,12939S), GLUT4 1:1000 (Abcam, ab33780), Insulin receptor substrate 1 1:1000(Cell Signaling Technology, 2383L), Ser636/639 Phospho-Insulin receptor substrate 1 (Cell Signaling Technology, 2388S). The western blot gels were quantified by evaluating the band intensities using ImageJ (Wang et al., 2017 ).

Analysis by SDS-PAGE was done on 4–20% Mini-protean precast gels (Bio-Rad Laboratories, Hercules, CA, USA) that were run in 25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3. Prior to electrophoresis, samples were incubated at 95°C for 5 min with an equal volume of 2xLaemmli sample buffer (Bio-Rad Laboratories), with or without 0.7 M β-mercaptoethanol. Page ruler (Thermo Fisher Scientific, Waltham, MA, USA) was used as molecular size marker.

These samples were processed according to [30 (link)]. Briefly, nanoparticles with an area of (1015 nm²) were incubated in the different exposure media (1) Leibowitz’s L15; (2) seawater) in a 1:52 ratio (area:µL incubation media). Then, nanoparticle–protein complexes were centrifuged through a sucrose cushion [42 (link)] and washed. The remaining volume was then eluted, and protein concentration as well as impurities (DNA, RNA, salts, solvents) were determined using a NanoDrop 3300 spectrophotometer (Thermo Fisher Scientific, Darmstadt, Germany). Samples were analyzed by 1D SDS-PAGE performed in a BioRad MiniProtean system using 4–20% MiniProtean Precast gels (Bio Rad. Hercules, CA, USA), followed by the Coomassie staining (Coomassie BrilliantBlue R250, Merck, Darmstadt, Germany) of the gels to determine the size of the proteins bound to the NPs.
For determining corona formation, nanoparticles were dispersed in exposure media, (1) Leibovit’z L-15 with 10% FBS and (2) filtered seawater, to create stocks with identical surface:volume ratios. Weathering conditions were aimed at investigating the adhesion of biomolecules to the NP solution, and thus solutions were incubated in falcon tubes with exposure media for 2 h and 2 weeks in an agitation platform before exposure of the cells (weathered nanoparticles).