Monolayers of host cells were lysed and denatured as previously described (Deka et al., 2006 (link)). The Western blot assays were conducted as described by Sun et al. (2008 (link)). The total protein concentration in cell lysates was normalized by analysis of a SYPRO Ruby stain (Bio-Rad) using a G-box (Bio-Rad) and SynGene software. Primary antibodies were anti-nectin-1 CK6 (sc-21722, Santa Cruz Biotechnology), anti-β-actin (MAB1501, Chemicon), anti-nectin-2 (AF2229, R&D systems), anti-HVEM (N-19) (sc-7766, Santa Cruz Biotechnology), anti-phospho-Akt (9271, Cell Signaling), anti-phospho-JAK (3331, Cell Signaling) anti-phospho-JNK (9251, Cell Signaling), anti-phospho-PI3K (4228, Cell Signaling) and anti-focal adhesion kinase c20 (FAK, sc-558, Santa Cruz). Primary antibody binding was detected with corresponding horseradish peroxidase-conjugated secondary antibodies and visualized using SuperSignal West Pico reagent (Pierce). Densitometry analysis was performed using a FX phosphorimager and Quantity One V2.5.0 software (Bio-Rad) or with a G-box and SynGene software (Biorad). To control for small variations in cell number and gel loading between sample lanes, the nectin-1 quantity in each sample was normalized to the amount of β-actin protein detected in that same lane (Sun et al., 2008 (link)).
G box
Manufactured by Bio-Rad
Sourced in Australia
The G-box is a compact and versatile imaging system designed for visualizing and analyzing a variety of samples, including DNA, RNA, and protein gels, as well as chemiluminescent and fluorescent Western blots. The system features a high-resolution camera, advanced imaging software, and a wide range of illumination options to capture high-quality images of your samples.
Automatically generated - may contain errors
Lab products found in correlation
Enhanced chemi luminescence (ecl), by Bio-Rad (2 mentions)
Fx phosphorimager, by Bio-Rad (1 mentions)
Anti phospho pi3k, by Cell Signaling Technology (1 mentions)
Sypro ruby stain, by Bio-Rad (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Mab1501, by Merck Group (1 mentions)
Anti phospho jnk, by Cell Signaling Technology (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Caspase 3, by Santa Cruz Biotechnology (1 mentions)
Lamin b, by Santa Cruz Biotechnology (1 mentions)
Anti β catenin antibody, by Cell Signaling Technology (1 mentions)
Sypro ruby, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Clarity western ecl kit, by Bio-Rad (1 mentions)
Biomix, by Meridian Bioscience (1 mentions)
Mmp 2, by R&D Systems (1 mentions)
Mmp 14, by R&D Systems (1 mentions)
9 protocols using g box
1
Protein Expression and Quantification
2
Quantification of Cytoplasmic and Nuclear Proteins
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Total protein content in the cytoplasmic and nuclear fractions was quantified with the Bradford method using BSA as a standard (Kruger, 2009 (link)). Samples (100 μg) were denatured using sample buffer at 95°C for 5 min, proteins were separated on 4–12% SDS-PAGE gel (Bio-Rad, Philadelphia, PA, United States) and electroblotted onto a nitrocellulose membrane (Schleicher & Schuell, Keene, NH, United States). Primary and secondary antibodies against Nrf2, caspase-3, β-actin and lamin-B (Santa Cruz Biotechnology, Santa Cruz, CA, United States) were used to detect corresponding proteins. The blot was developed using ECL (Bio-Rad, Philadelphia, PA, United States) and the signals captured using a gel documentation system (GBOX, Syngene, United Kingdom).
3
β-catenin Accumulation Analysis
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Infected IK and SHT-290 monolayers from IK/SHT-290 co-cultures were collected in 8 M urea. The protein samples were examined by SDS-PAGE electrophoresis for Sypro Ruby (BioRad) staining and Western Blot analysis as previously described (Deka et al., 2006 (link)). Blots were probed with anti-β-catenin antibodies (Cell Signaling). Protein accumulation in each experiment was quantified from triplicate samples using a BioRad G-box and SynGene software. β-catenin accumulation was normalized to the total amount of protein detected in each sample by Sypro Ruby staining.
4
Quantitative Immunoblot Analysis of Insulin Signaling
5
Western Blot Analysis of Flii in Diabetes
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Western blot analysis of Flii protein expression was carried out on mouse plasma from healthy and diabetic mice, as previously described [22 (link)]. SDS-PAGE loading buffer (3 μL of 25 nM Tris, pH 6.8; 8% glycerol; 1% SDS; 0.02% bromophenol blue; and 5% 2-mercaptoethanol) was added to 15 μL of each protein sample. Samples were boiled at 100 °C for 5 min and then vortexed. Samples (15 μL) were separated using a 10% polyacrylamide gel (sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)). Electrophoresis was performed in 1× Western Running Buffer (25 mM Tris, glycine, 192 mM, 0.1% SDS) at 25 mA per gel, and transfer was carried out at 250 mA for 1 h at 4 °C. The membrane was blocked in 15% skim milk in PBS for 1 h and blotted with primary antibodies against Flii (1:200) in 6% skim milk overnight at 4 °C. The membrane was washed 3 times for 5 min in 1× TBST (TBS, 0.1% Tween20) and probed with secondary antibodies (1:2000) for 1 h at room temperature. The membrane was washed 2 times, developed using a Clarity Western ECL kit (Bio-Rad, Gladesville, NSW, Australia), and imaged under UV light using a SynGene G-Box. Results were representative of three independent experiments.
6
Gene-specific PCR protocol for cDNA analysis
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Gene-specific PCRs were performed with Biomix® (Bioline, Sydney, Australia) using the primer sequences shown in Supplementary Table 2 . Cycling was initiated by denaturation (94°C, 5 min) followed by 25–33 cycles that involved denaturation (94°C, 30 s), annealing (0.5–1 min) and elongation (72°C, 1 min) then terminated by a final extension step (72°C, 10 min). Amplified cDNA mixed with 5x loading dye (Bioline, Australia) was resolved on 1% w/v agarose gels containing ethidium bromide (2 μg/mL). Products were visualized and imaged under short-wavelength UV light in a G-Box (BioRad, Australia) and converted to TIF files using standard imaging software. Semi-quantitative densitometry was carried out using Image J software (v1.42) made available in the public domain from the NIH, USA (39 (link)).
7
RNA Folding Dynamics with Metal Ions
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Purified RNA (2 μg) was heated at 90°C for 2 min followed by incubation at room temperature for 3 min, then subsequently allowed to refold in 50 mM HEPES [pH 7.5], 50 mM NaCl at room temperature for 10 min. Various divalent metal ion concentrations ranging from 0.01 to 5 mM were added and RNA mixture was left at room temperature for an additional 30 min prior to mixing with loading dye (10% glycerol and 0.01% xylene cyanol). RNA was separated by native PAGE (8% gel prepared with 19:1 acrylamide/bisacrylamide) at 4°C in running buffer containing 34 mM Tris, 66 mM HEPES [pH 7.5], and 3 mM MgCl2, which was recirculated every hour. RNA was stained with ethidium bromide and observed with a Gbox (Biorad).
8
Western Blot Analysis of MMP-2 and MMP-14
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40 μg of protein from each sample were loaded onto 10% SDS-PAGE gels and after electrophoretic separation were transferred to nitrocellulose membranes. The transfer was confirmed by staining the membrane with 0.5% Ponceau red solution. Immunoblotting followed standard methods which were briefly as follows (B). The washed membranes were blocked with 5% (w/v) fat free milk for 1hr and after removal of excess blocking solution by washing, the membranes were incubated overnight with the primary antibodies (MMP-2 (1:2000) R&D Systems, USA; MMP-14 (1:2000) R&D Systems, USA; actin (1:3000) Abcam, Cambridge, MA, USA). The membranes were washed and incubated with secondary antibodies for 2 h at room temperature (anti-goat-HRP (MMP-2), anti-mouse-HRP (MMP-14), anti-rabbit-HRP (actin) (1:2000)). Finally, the membranes were and developed with enhanced chemiluminescence (ECL- Bio-Rad Laboratories Inc., São Paulo, Brazil) in a Syngene G-BOX with its GeneSys program. The ImageJ program was used to determine relative band intensities.
9
Caspase-1 Protein Immunoblotting
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Supernatants were concentrated x10 using Amicon Ultra centrifugal units (Merck Millipore, Darmstadt, Germany). The protein content of lysates was analysed using a BCA protein assay kit (Life Technologies; Paisley, UK) according to manufacturer's instructions. Supernatants and lysates were loaded with sample reducing agent and LDS sample buffer (Invitrogen, Novex, Fischer scientific, UK) in 10 μl, loaded onto a 12% acrylamide gel and separated proteins transferred onto a PVDF membrane (Bio-Rad, Hercules, California, US). Membranes were washed in 1X TBS, 1% Tween (TBST) and blocked in 1X TBST containing 5% Milk powder for 1 hour at room temperature. Anti-caspase-1 (Caspase-1 Polyclonal Antibody, NBP1-45433, Novus biologicals, Bio-Techne Ltd Abingdon, UK) was added according to manufacturer's recommendations in 5-10 ml of 1X TBST 5% milk at 4 o C overnight. Membranes were incubated with HRP-conjugated goat anti-rabbit IgG, diluted to 1:5,000 in 1X TBST 5% milk powder for 1 hour before visualisation with Clarity™ Western ECL Substrate (Bio-Rad, Hercules, California, US) using a Syngene G: BOX with GeneSys software.
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