Criterion tgx precast gel

A549 cells were seeded in a 60-mm plate at 80% confluency. The following day, cells were transfected with 1 μg of plasmid DNA (psi-miR-34 WT or psi-miR-34 MT) and 200 pmol ON-TARGETplus siRNA SmartPool (Dharmacon) using Lipofectamine 2000 as indicated. After transfection, the cells were re-plated in a six-well plate at 50% confluency. After a 12-h incubation, the cells were exposed to 2 Gy of IR. At 4, 12 and 24 h post IR, cells were washed with PBS and lysed in wells with Passive Lysis Buffer (Promega). 25 μl of lysate was withdrawn for luciferase analysis (described above); 75 μl of lysate was boiled in SDS Sample buffer and samples were separated using a 4–20% Criterion TGX Precast Gel (Bio-Rad). Protein was transferred to Whatman BA85 Nitrocellulose and protein was detected using: anti-ATM 2C1 (1A1) (ab78, Abcam, 1:1,000), anti-Clp1 N3C3 (GTX115518, GeneTex, 1:1,000) and sheep anti-mouse IgG, HRP-linked Ab (NA931, GE Healthcare, 1:50,000) and HRP-conjugated anti-rabbit (ab6721, Abcam, 1:25,000). Pierce ECL western blotting substrate (PI-32109) was used for detection.

Snap-frozen kidney pieces were homogenized in CellLytic^TM MT Cell Lysis reagent (Sigma-Aldrich, St. Louis, MO, USA) supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Fisher, Waltham, MA, USA). Protein concentration was determined with a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). 35 µg of homogenized protein were prepared/well in 2x Laemmli buffer (Bio-Rad laboratories, Hercules, CA, USA). Tissue lysates were run on a 4–15% Criterion TGX precast gel (Bio-Rad Laboratories). Membranes were incubated with primary antibody overnight at 4 °C as follows: KIM-1 (1:1500 or 0.13 µg/mL, R&D Systems, Minneapolis, MN, USA), NGAL (1:1500 or 0.13 µg/mL, R&D Systems, Minneapolis, MN, USA), total eNOS (1:1000 or 0.25 µg/mL, BD Biosciences, Franklin Lakes, NJ, USA), p1177 eNOS (1:1000 or 0.25 µg/mL, BD Biosciences, Franklin Lakes, NJ). Secondary antibodies were applied the next day for 1 h at room temperature (1:10,000 KPLaboratories, Gaithersburg, MD, USA) and blots were developed using Clarity™ Western Blotting Substrate (Bio-Rad Laboratories). A ChemiDoc™ Touch Imaging System was used for exposure (Bio-Rad Laboratories). Densitometry was performed in Image J software [38 ], and bands of interest were normalized to total protein as determined by a simultaneously run Coomassie stained gel.

To perform western immunoblotting, cells were lysed using RIPA lysis buffer (Thermo Scientific) and protein concentrations were assessed using a modified Lowry method (DC Protein Assay; Bio-Rad, Hercules, CA, USA). Cell lysates were loaded onto a 4–15% Criterion TGX Precast gel (Bio-Rad) and protein were transferred onto an Immobilon-P PVDF membrane (EMD Millipore, Burlington, MA, USA). The membranes were blocked with 5% (wt/v) non-fat milk/PBS-Tween20 (Sigma-Aldrich) solution and incubated with primary antibodies overnight at 4 °C (Supplementary Table S3). As secondary antibodies, anti-mouse or anti-rabbit horseradish peroxidase polymer antibodies were used (GE Healthcare, Chicago, IL, USA). Beta-actin served as a loading control. Bands were visualized using Amersham enhanced chemiluminescence detection reagents (GE Healthcare). All full-length original western immunoblots used in the study are provided in Supplementary material 2.

The frozen kidney samples were homogenized in lysis buffer (20 mM Na-HEPES [pH 7.5], 100 mM NaCl, 1% Triton X-100, 15 mM NaF, 1 mM Na₃VO₄, 10 mM Na₄P₂O₇, 1 mM EDTA, Protease Inhibitor Cocktail ([Roche, Tokyo, Japan], and Phosphatase Inhibitor Cocktail 2 [Sigma-Aldrich]) on ice and centrifuged at 5,591×g for 10 minutes to remove debris. The protein concentrations in the supernatants were determined using 5 μg/μL. Ten micrograms of protein from each specimen were suspended in loading buffer, separated on a 4–15% Criterion^™ TGX^™ Precast Gel (Bio-Rad, Tokyo, Japan), and electrophoretically transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in TBS and incubated with a 500-fold dilution of the primary antibodies overnight at 4°C. After three 5-minute washes with washing buffer (0.1% Tween 20 in TBS), the membranes were incubated with the secondary antibodies for 1 hour at room temperature. After three 10-minute washes with washing buffer, the membrane was incubated with Enhanced Chemiluminescence (ECL) Western Blotting Detection Reagents (GE Healthcare, Tokyo, Japan) and exposed using LuminoGraph (ATTO, Tokyo, Japan) and ImageSaver6 (ATTO). GAPDH was used as an internal control.

Total protein was extracted from liver and fat using RIPA buffer supplemented with protease inhibitor cocktail (cOmplete from Millipore-Sigma) with a tissue homogenizer (2X 30 s at 6000 rpm). Then samples were incubated with gentle agitation for 30 min at 4 degrees, followed by centrifugation for 20 min at 13 000 rpm. 5 ug protein lysates were run on 4–15% Criterion TGX Precast Gel (Bio-Rad, Denmark). Proteins were transferred to a nitrocellulose membrane (Hybond ECL, GE Healthcare, UK). After blocking (5% BSA at room temperature for one hour), the membranes were incubated overnight at 4 °C with the following primary antibodies: Anti-Acetyl-Coenzyme Carboxylase antibody (EP687Y) (ab45174), and Anti-Fatty Acid Synthase antibody (ab22759), both 1:4000 dilution from Abcam. The membranes were incubated with goat anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature (Dako, P0448 diluted 1:4000) and afterward, the antigen–antibody complex was visualized using an enhanced chemiluminescence system (ECL, Amersham ECL Plus, GE Healthcare). All Western blots were normalized to total protein, measured using Stain-Free technology, a method previously validated by others^{35 (link),36 (link)}. The total protein gel image can be found in Supplementary information S1, Fig. 2.

Renal cortex proteins were lysated in a dissecting buffer (0.3 M sucrose, 25 mM imidazole, and 1 mM (EDTA), pH 7.2) including protease inhibitors Complete Mini Protease Inhibitor Cocktail Tablets (Roche Diagnostics, Hvidovre, Denmark) and Phosphatase Inhibitor Cocktail 2 and 3 (Sigma-Aldrich, Brøndby, Denmark) using a tissue homogenizer (Qiagen, Hilden, Germany) followed by centrifugation. The total protein concentration was determined using a Pierce BCA protein assay kit (Roche Diagnostics, Hvidovre, Denmark) following the manufacturer’s instructions.
Proteins were separated on a 12% Criterion TGX Precast Gel (Bio-Rad Laboratories, Copenhagen, Denmark) and transferred to a Hybond ECL nitrocellulose membrane (GE Healthcare, Hatfield, UK). The membrane was then blocked in 5% non-fat dry milk in PBS-T (80 mM Na₂HPO₄, 20 mM NaH₂PO₄, 100 mM NaCl, and 0.1 Tween 20, pH 7.4), washed in PBS-T, and incubated with primary antibodies overnight at 4 °C. Subsequently, the membrane was again washed and incubated with HRP-conjugated secondary antibody for one hour at room temperature. Antigen-antibody reactions were visualized using a chemiluminescence system (Amersham ECL Plus, GE Healthcare). All western blots were normalized to total protein content measured with Stain-Free technology [23 (link)]. Primary and secondary antibodies are listed in Table 1.