Flash-frozen mouse brain areas were homogenised by sonication in 2× sample buffer (Pierce LDS Sample Buffer; Thermo Fisher Scientific) containing 0.2 M DTT. Lysates were then centrifuged at 13,000g for 15 min, and supernatants were removed and denatured at 70°C for 10 min. Neurons and HEK293T cells treated with Bafilomycin A1 (Sigma-Aldrich) were lysed directly in 2× sample buffer containing 0.2 M DTT, followed by sonication and incubation at 70°C for 10 min. All lysed samples were run on NuPAGE 4–12% Bis-Tris polyacrylamide gels (Thermo Fisher Scientific) and transferred to a polyvinylidene difluoride membrane (Millipore) using wet transfer. After blocking in 5% non-fat milk in TBST for 1 h, the membranes were incubated with primary antibodies overnight at 4°C or at RT for 1–2h. Peroxidase-conjugated (HRP) secondary antibody incubation was carried out at RT for 1 h. For Western blot detection, the membranes were incubated with ECL (Amersham ECL Prime Western Blotting Detection Reagent) and visualised with a chemiluminescence digital imaging system (Amersham Imager 600 RGB) or developed using film (Amersham Hyperfilm ECL; Western blots in Fig S1B only).
Imager 600rgb
Manufactured by Cytiva
The Imager 600RGB is a compact, multi-function imaging system designed for life science applications. It offers high-resolution imaging capabilities, supporting a range of imaging techniques including fluorescence, chemiluminescence, and colorimetric detection. The system features intuitive software for image capture and analysis, providing users with a streamlined workflow.
Automatically generated - may contain errors
Lab products found in correlation
Pierce lds sample buffer, by Thermo Fisher Scientific (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Nupage 4 12 bis tris polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Hyperfilm ecl, by Cytiva (2 mentions)
Bafilomycin a1, by Merck Group (2 mentions)
P nitrobenzyl mesylate, by Abcam (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Amersham ecl prime western blotting detection reagent, by Cytiva (1 mentions)
Anti phospho pkc substrate antibody, by Cell Signaling Technology (1 mentions)
Anti α tubulin antibody, by Merck Group (1 mentions)
6 benzyl atpγs, by Biolog (1 mentions)
Mouse anti flag m2 antibody, by Merck Group (1 mentions)
Horseradish peroxidase conjugated anti mouse, by Merck Group (1 mentions)
Optitran ba s83, by Cytiva (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Reverse transcription kit, by Qiagen (1 mentions)
Na green, by Beyotime (1 mentions)
Nupage novex bis tris gel, by Thermo Fisher Scientific (1 mentions)
Ecl prime western blotting detection reagent, by Cytiva (1 mentions)
13 protocols using imager 600rgb
1
Western Blot Analysis of Brain and Cell Lysates
2
PACAP Regulation of PKA and PKC Substrates
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aNPCs were dissociated with Accutase and plated on poly-l-lysine- and laminin-coated 24-well plates at 5 × 104 cells per well. They were allowed to grow in the presence or absence of PACAP for 4 days and then washed with PBS and lysed with RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2% Igepal CA-630, 0.25% sodium deoxycholate, 1 mM NaF, 1 × SigmaFAST protease inhibitor cocktail, and 1 mM DTT). Protein concentration was measured using the BCA assay and equal amounts of protein were loaded onto an SDS-PAGE gel. Following transfer, nitrocellulose membranes were blocked with the blocking buffer containing 5% bovine serum albumin in tris-buffered saline + 0.05% Tween-20 and incubated overnight at 4℃ with anti-phospho-protein kinase A (PKA) substrate antibody (Cell Signaling cat. #9624), anti-phospho-PKC substrate antibody (Cell Signaling cat. #2261), or anti-α-tubulin antibody (Sigma cat. T6199) diluted to 1 µg/mL in the blocking buffer, followed by washes and incubation with HRP-conjugated secondary antibodies. Chemiluminescent signal was detected using the Amersham Imager 600RGB and densitometric measurements were performed in ImageJ.
3
High-Resolution Carbohydrate Electrophoresis
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Gels were prepared
using a 50 mL
solution of 20% acrylamide in TBE buffer (89 mM Tris, 89 mM borate,
2 mM EDTA, pH 8.3) containing 7 M urea. Polymerization was initiated
by the addition of 162 μL of (NH4)2S2O8 (25% in water) and 44 μL of N,N,N′,N′-tetramethylethylenediamine. The gels were of the 8- or 17-well format
(width 20 cm) with 30 cm well-to-read length and 0.75 mm thickness.
The running buffer was TBE. Electrophoresis was performed at a constant
power of 35W (Consort EV3330) and ambient temperature with cooling
by air (ventilator); the front glass was covered with an external
aluminum plate. After prerunning for 30 min, the wells were thoroughly
rinsed with TBE, and small volumes (20–50 μL, ca. 50%
formamide) of the samples were loaded. One gel lane between two samples
was usually left free to avoid cross-contamination and ease the lane
tracking. The electrophoresis voltage during separation was 1700–2200
V, and the analysis was run untilAPTS reached the bottom
of the gel (1.5–2 h). The bands of labeled carbohydrates were
detected by emission in a UV viewing cabinet (254/365 nm) equipped
with a digital camera or using Amersham Imager 600RGB.
using a 50 mL
solution of 20% acrylamide in TBE buffer (89 mM Tris, 89 mM borate,
2 mM EDTA, pH 8.3) containing 7 M urea. Polymerization was initiated
by the addition of 162 μL of (NH4)2S2O8 (25% in water) and 44 μL of N,N,N′,N′-tetramethylethylenediamine. The gels were of the 8- or 17-well format
(width 20 cm) with 30 cm well-to-read length and 0.75 mm thickness.
The running buffer was TBE. Electrophoresis was performed at a constant
power of 35W (Consort EV3330) and ambient temperature with cooling
by air (ventilator); the front glass was covered with an external
aluminum plate. After prerunning for 30 min, the wells were thoroughly
rinsed with TBE, and small volumes (20–50 μL, ca. 50%
formamide) of the samples were loaded. One gel lane between two samples
was usually left free to avoid cross-contamination and ease the lane
tracking. The electrophoresis voltage during separation was 1700–2200
V, and the analysis was run until
of the gel (1.5–2 h). The bands of labeled carbohydrates were
detected by emission in a UV viewing cabinet (254/365 nm) equipped
with a digital camera or using Amersham Imager 600RGB.
4
Quantifying FtsZ and ZapA Levels in Bacterial Cells
5
Quantifying Protein Concentrations for Kinase Assays
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To establish the concentration of the proteins needed for kinase assays, purified proteins were separated via gel electrophoresis together with known amounts of BSA. The gel was stained with Coomassie stain and imaged using colorimetric assessment on a digital imaging system (Amersham Imager 600RGB). The intensity of the bands was quantified and the concentration of Raph1 and NDR1 constructs established based on the known amounts of BSA loaded onto the same gel.
For the kinase assay, 500 ng Raph1 was incubated with 50 ng NDR1 in buffer containing 20 mM Tris–HCl, pH 7.5, 10 mM MgCl2, l μM okadaic acid, 1 mM DTT, 1× protease inhibitor cocktail (Roche), 100 μM ATP and 0.5 mM 6-benzyl-ATPγS (Biolog) at 30°C, with rotation, for 30 min. The reactions were quenched with 20 mM EDTA and alkylated with 5 mM p-nitrobenzyl mesylate (Abcam) for 30 min at room temperature. The proteins were solubilised in sample buffer (Pierce LDS Sample Buffer; Thermo Fisher Scientific) containing 0.1 M DTT and denatured by incubation at 70°C for 10 min, in preparation for Western blot analysis.
For the kinase assay, 500 ng Raph1 was incubated with 50 ng NDR1 in buffer containing 20 mM Tris–HCl, pH 7.5, 10 mM MgCl2, l μM okadaic acid, 1 mM DTT, 1× protease inhibitor cocktail (Roche), 100 μM ATP and 0.5 mM 6-benzyl-ATPγS (Biolog) at 30°C, with rotation, for 30 min. The reactions were quenched with 20 mM EDTA and alkylated with 5 mM p-nitrobenzyl mesylate (Abcam) for 30 min at room temperature. The proteins were solubilised in sample buffer (Pierce LDS Sample Buffer; Thermo Fisher Scientific) containing 0.1 M DTT and denatured by incubation at 70°C for 10 min, in preparation for Western blot analysis.
6
Protein Analysis by Western Blotting
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Protein samples were separated on a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then electroblotted onto nitrocellulose membranes (Whatman Optitran BA-S 83), blocked with 5% non-fat dry milk in Tris-buffered saline + 0.1% Tween (TBS-T). Probing of the blots was done with monoclonal ANTI-FLAG M2 Antibody (mouse) from Sigma (F1804) 1 µl diluted 1000× in 5 ml of TBS-T buffer and 5% non-fat dry milk, followed by incubation with 1 µl horseradish peroxidase-conjugated anti-mouse (Sigma) for 2 h in 10 ml (1/10,000) of TBS-T and 5% non-fat dry milk. Protein signals were detected with chemiluminescence imaging (Amersham Imager 600RGB).
7
Quantitative Real-Time RT-PCR Assay for Gene Expression
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Total RNA was isolated from control and TNF-α treated HL-1 cells or TNFαR1/R2-DKO mouse heart using Qiagen RNeasy Mini kit and 1.25 µg RNA was processed for cDNA synthesis using Qiagen reverse transcription kit (205,311) as per the supplier instructions. An aliquot of cDNA template (25–50 ng), 10 μl of QuantiFast SYBR green master mix (204,054), and appropriate primers were used for quantitative real-time RT-PCR (qPCR) analysis and analyzed in a Light Cycler (Roche Bio) [49] (link). Fold changes of mRNA expression of different targets were quantified using Ct values, and the relative mRNA expression levels for all samples were obtained by normalizing to the level of the housekeeping gene Arbp1 or Gapdh mRNA expression. All the primers used for qPCR were given in Table 1 . Semi-quantitative PCR was also carried out for TNFR1, TNFR2 and GAPDH using gene specific primer by following PCR conditions; an initial denaturation at 95 °C for 3 min followed by 95 °C for 30 s, 60 °C for 30 s, 72 °C for 45 s, and finally an extension at 72 °C for 5 min. The PCR products were run in 2% agarose gel and the products were seen at −100 bp sizes by ethidium bromide staining [50] (link) using Amersham Imager 600 RGB.
8
Agarose Gel Electrophoresis of (SA)2-Peptide-pDNA Complexation
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Agarose gel electrophoresis was conducted to study the complete complexation of (SA)2-peptide with pDNA. Nanoparticles were prepared with (SA)2-peptide and pDNA at ratios of 5:1, 10:1, 15:1, 20:1, or 25:1. Ten microliters of solution containing 0.3 μg of pDNA was mixed with DNA loading buffer and loaded into parallel wells of a 1.2% (w/v) agarose gel containing 0.5 μl ml−1 NA-Green (Beyotime) in tris-acetate-EDTA buffer to evaluate the DNA-complexation effect of the (SA)2-peptide compared with that of naked pDNA. Electrophoresis was performed at 90 V for 60 min, and gel retardation was visualized by using an Amersham Imager 600 RGB.
9
Labeling Proteins with Fluorescent Dye
10
Quantifying Tight Junction Protein Expression
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Cell lysates were prepared from cell populations maintained under the conditions used for the measurement of paracellular permeability. Total cell protein lysates were prepared as previously described (Caswell et al., 2013 (link)). Western blotting was performed as previously described (Caswell et al., 2013 (link)). Primary antibody dilutions for western blotting were as follows: ZO‐1—1:500–1:2,000; ZO‐2—1:500–1:2,000. HRP‐conjugated anti‐rabbit Fc fragment antibodies were used at a dilution of 1:10,000–1:20,000. Signals were developed using the Pierce West Pico SuperSignal Reagent. Chemiluminescence signals were imaged using an Amersham Imager 600RGB. Presented blots are representative of at least four separate blots obtained from at least four independent sets of samples.
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