Digital imaging system

Lysates from cells were made using mammalian protein extraction reagent with protease and phosphatase inhibitors diluted 1:100 (Thermo Fisher Scientific, Waltham, MA, USA). Proteins were separated using SDS‐PAGE. Target proteins were detected with the appropriate antibodies and images were captured using a digital imaging system (Bio‐Rad, Hercules, CA, USA). All bands were observed around the size specified in supplementary material, Table S2A. Densitometric quantitation of target proteins was calculated using ImageJ relative to loading controls β‐actin or GAPDH depending on the target protein size (see supplementary material, Table S2B for data).

Genomic DNA was isolated from each sirloin meat sample at room temperature using a PureLink^™ Genomic DNA Mini Kit (Invitrogen Life Technologies, USA) following protocol provided by the manufacturer. In brief, 20-25 mg of fresh meats were minced, immersed in digestion buffer mix, and incubated at 55°C for 2 h with occasional vortexing. After spinning for 3 min at 16,000× g, supernatant was mixed with 20 μL of RNase A and incubated for 2 min. The lysate was mixed with 200 μL of genomic lysis/binding buffer, added with 200 μL of absolute ethanol, and mixed by short vortexing. The entire mixture was transferred to a spin column in a collection tube and spun at 1000× g for 1 min. After replacing the collection tube, 500 μL of wash buffer-1 was added, and the column was respun at 10,000× g for 1 min. This washing step was repeated using wash buffer-2 and 3-min spinning at 16,000× g. Genomic DNA was eluted from the spin column by 1 min incubation with 50 μL of elution buffers followed by spinning at 16,000× g for 1 min. The quality of DNA extract was checked by electrophoresis on a 1% agarose/1×TAE gel stained with a SYBR™ Safe (Invitrogen Life Technologies, USA) stain using a 100-bp ladder as a molecular size marker, and visualized using a digital imaging system (Bio-Rad, USA). Purified DNA extract was stored at −20°C [23 ].

This commercially-available immunoassay (Cell Signaling Technology, Danvers, MA) was used according to the manufacturer's instructions. Briefly, cell lysates were harvested after 24 h of mono and co-culture using the buffer provided with the kit. The Array Blocking Buffer was added to each well and incubated for 15 min at room temperature. Subsequently, the lysate was added to each well and incubated overnight at 4 °C. After washing, the detection antibody cocktail was added to each well and incubated for 1 h at room temperature. Horseradish peroxidase (HRP)-linked streptavidin was added to each well and incubated for 30 min at RT. The slide was then covered with Clarity™ ECL (Bio-Rad) and images were captured using a digital imaging system (Bio-Rad). Image J version 1.46r (NIH, http://imagej.nih.gov/) was used for spot quantifications.

After lysing cells with RIPA (Beyotime, Shanghai, China) to obtain protein samples, protein concentration was measured using the BCA method. After electrophoresis, 5% skimmed milk was blocked for 2 h at room temperature, the primary antibody was incubated overnight at 4 °C, and the secondary antibody was incubated for 1 h at room temperature; anti-p38/p-p38, anti-ERK/p-ERK, anti-JNK/p-JNK, anti-p65/p-p65, anti-IKKα/p-IKKα, anti-IκBα/p-IκBα, anti-Sirt1, anti-β-Actin, anti-β-Catenin were obtained from Cell Signalling Technology (1:1000 dilution); anti-PGC1α, anti-COX15, anti-NDUFV2, anti-ATP5D, anti-ATP5H were obtained from Proteintech Biotechnology (1:1000 dilution); anti-TFAM was obtained from Absin Bioscience (1:1000 dilution); anti-p-GSK3β/GSK3β was purchased from Santa Cruz Biotechnology (1:1000 dilution); Peroxidase-conjugated secondary antibody (1:4000 dilution) was used, and ECL reagent (Beyotime, Shinghai, China) was added at a 1:1 ratio to develop the image on a digital imaging system (Bio-Rad, USA).

After the kidneys were collected from the mice, an equivalent of protein was resolved on dodecyl sulfate (SDS)-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes. The antibodies used are shown below: AGEs and RAGE antibodies were from Abcam, UK; p-IKKα/β, IKKβ, IKKα, p-IκBα, IκBα and NF-κB p65 antibodies were from Cell Signaling Technology, USA; p-NF-κB p65 antibody was from Santa Cruz Biotechnology, Glostrup, Denmark and GAPDH antibody was from Beijing Zhong Shan Golden Bridge Biotechnology Co., Ltd., China, with a dilution of 1:1000. Horseradish peroxidase-labeled secondary antibody was from Beijing Zhong Shan Golden Bridge Biotechnology Co., Ltd., with a dilution of 1:10,000. The membranes were developed with enhanced chemiluminescence (Thermo Scientific, USA) and visualized using a digital imaging system (BIO-RAD Laboratories, Inc., USA).

Protein samples from total cell lysates (20 or 50 μg) were subjected to electrophoretic separation on a 7.5 or 10% polyacrylamide gel and transblotted onto a polyvinylidine fluoride (PVDF) membrane (Millipore). Blots were blocked at room temperature for 1 hour in Tris-buffered saline (TBS)/Tween 20 (TBS-T) (0.05%) containing 5% bovine serum albumin (BSA) and incubated with: 1:500 rabbit polyclonal anti-EGFR (#2235); 1:500 rabbit monoclonal anti-phospho-EGFR (Tyr1173) (#4407); 1:500 rabbit monoclonal anti-phospho-EGFR (Tyr1068) (#3777); 1:1000 mouse monoclonal antibody anti-phospho-p44/42 MAPK (ERK1/2) (#9106); 1:1000 rabbit polyclonal anti-p44/42 MAPK (ERK1/2) (#9102); 1:1000 rabbit polyclonal anti-phospho-Akt (Thr308) (#9275); 1:1000 rabbit polyclonal anti-Akt (#9272) (Cell Signaling Technology); 1:10000 mouse monoclonal anti-actin (clone C4) (MP Biomedicals). Blots were subsequently washed and incubated with HRP-anti-mouse or HRP-anti-rabbit antibodies at 1:1500 (Dako). Detection was performed using Lumi-Light Western blotting substrate (Roche Diagnostics Nederland B.V.). Images were captured using a digital imaging system (Bio-Rad).

The amplified products were then separated by agarose gel electrophoresis. PCR-products were run on 1% agarose gels (HiMedia, Mumbai, India) containing Ethidium Bromide stain (EtBr) (HiMedia, Mumbai, India) with 1x TAE buffer (40 mM Tris- HCl, 20 mM Naacetate, 1mM EDTA, pH 8.4) and the bands were visualized under an UV transilluminator (Biometra, Germany). Images were captured with digital imaging system (Bio-Rad).