Membrane proteins were biotinylated and purified using the Pierce Cell Surface Protein Isolation Kit according to the manufacturer’s instructions (Pierce, Thermo Scientific, Rockford, IL, USA). Western blot analysis was performed using a mouse monoclonal primary antibody against V5 (Invitrogen) and then a polyclonal goat anti-mouse immunoglobulin/HRP (Dako) as secondary antibody. The membranes were incubated with the Immobilon Forte Western HRP substrate according to the manufacturer’s instructions (Merck Millipore, Burlington, MA, USA) and digitized for pattern analysis using the GeneGnome system (Syngene), Paris, France).
Genegnome system
Manufactured by Syngene
Sourced in United Kingdom
The GeneGnome system is a high-performance imaging system designed for molecular biology applications. It provides sensitive detection and quantification of chemiluminescent, fluorescent, and colorimetric signals from a variety of sample types, including Western blots, nucleic acid gels, and protein gels. The system features a cooled CCD camera, interchangeable excitation and emission filters, and advanced image analysis software to enable accurate and reproducible data analysis.
Automatically generated - may contain errors
Lab products found in correlation
Polyclonal goat anti mouse immunoglobulin hrp, by Agilent Technologies (1 mentions)
Immobilon forte western hrp substrate, by Merck Group (1 mentions)
Enhanced chemiluminescence system, by GE Healthcare (1 mentions)
Grp78, by Cell Signaling Technology (1 mentions)
P ire1α, by Cell Signaling Technology (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Ire1α, by Cell Signaling Technology (1 mentions)
Nf κb pathway sampler kit, by Cell Signaling Technology (1 mentions)
P eif2α, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Gapdh, by Proteintech (1 mentions)
Protein a g agarose, by Santa Cruz Biotechnology (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene fluoride (pvdf), by Cytiva (1 mentions)
Pierce ecl plus, by Thermo Fisher Scientific (1 mentions)
O8264, by Merck Group (1 mentions)
Phospho nf κb p65, by Cell Signaling Technology (1 mentions)
Genesys software, by Syngene (1 mentions)
7 protocols using genegnome system
1
Membrane Protein Biotinylation and Western Blot
2
Western Blot Analysis of Protein Markers
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Total proteins were extracted from hippocampus tissues or NSCs using RIPA lysis buffer (Beyotime) supplemented with phosphatase and protease inhibitors and PMSF, and then quantified by BCA assay (Meilunbio). Western blot was performed as previously described.[24 ] GRP78 (#3711; 1:1000), PERK (#3192; 1:1000), p‐eIF2α (#9721; 1:1000), CHOP (#2895; 1:1000), IRE1α (#3294; 1:1000), p‐IRE1α (#9721S, 1:1000) and NF‐κB pathway sampler kit (#9936) were from Cell Signaling Technology; ATF6 (#37149, 1:1000) was from Abcam. β‐actin (MA5‐15739, 1:5000) was from Invitrogen; GAPDH (#60004, 1:5000) was from Proteintech. The horseradish peroxidase‐conjugated anti‐rabbit IgG (SA00013‐3, Proteintech) or anti‐mouse IgG (SA00001‐2, Proteintech) secondary antibodies were used, followed by detection with the enhanced chemiluminescence system (GE Healthcare). ECL Western blot analysis system was utilized for detection of the immunoreactive bands according to the manufacturer's instructions by using the GeneGnome system (Syngene). β‐actin or GAPDH was used as a loading control.
3
Immunoblotting and Co-immunoprecipitation of Adenosine Receptors
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Western blot: proteins were extracted from hypothalamic tissues, separated by SDS-PAGE, and transferred to PVDF membranes. The membranes were then blocked by 5% non-fat milk and incubated with rabbit anti-A1R (1:1,000), anti-A2AR (1:1,000), anti-A2BR (1:1,000) or anti-A3R (1:500) antibody, or mouse anti-β-Actin antibody (1:2,000). After incubation with horseradish peroxidase labelled secondary antibody (1:4,000), the membranes were exposed to the Supersignal West Femto Maximum Sensitivity Substrate (Thermo Fisher, Waltham, MA). Chemiluminescence was recorded with the GeneGnome system (Syngene, Cambridge, UK). Co-immunoprecipitation: HEK293T cells were transfected with pcDNA3 HA-A1R and Myc-OTR (Origene, Rockville, MD) expressing plasmids. Cell lysates were incubated with 2 μg of non-immune IgG, anti-HA or anti-Myc antibody. Immunoprecipitates were prepared by incubation with protein A/G-agarose (Santa Cruz Technology, Santa Cruz, CA), and subjected to western blot with either anti-Myc or anti-HA antibody. Uncropped blot images with molecular weight reference are shown in Supplementary Fig. 16 through 18.
4
Western Blot Analysis of Signaling Proteins
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Cells were lysed in PBS containing 2% NP-40 and lysates were subjected to SDS-PAGE (BioRad Miniprotein) and proteins were blotted onto polyvinylidene difluoride membrane (PVDF, Amersham Pharmacia Biotech). The primary antibodies anti-NF-κB p65 (#sc-8008, dilution 1/500) from Santa Cruz Biotech, phospho-NF-κBp65 (#3033, dilution 1/500) from Cell signaling, TRPC1: rabbit anti-TRPC1 (1:500, T8276; Sigma-Aldrich, United States), STIM1: rabbit anti-STIM1 (1:500, 4916S; Cell Signaling, United States) and Orai1: rabbit anti-Orai1(1:250, O8264; Sigma-Aldrich, United States) were revealed by HPR-conjugated secondary antibodies (Amersham Pharmacia Biotech) and enhanced chemiluminescence (Pierce ECL Plus, Thermo Scientific) according to the manufacturer’s instructions. Images were acquired with a Genegnome system and GeneSyssoftware (Syngene) (17 (link)–19 (link)).
5
Protein Isolation and Western Blotting
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Protein isolation and western blotting procedures have been described before.14 (link) Whole cell lysates from cell cultures were prepared by adding lysis buffer (20 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1 mM EDTA, pH 8; 0.1% NP40; 0.01% SDS with protease inhibitor cocktail and phos-STOP (Roche, Germany).
For xenograft tumour lysate preparation, excised, snap frozen tumours were homogenized in above mentioned lysis buffer using Tissue Grinder, sonicated (50% amplitude for 2 min with 10 s on and 20 s off pulses) and lysates were cleared by centrifugation.
Total proteins isolated were quantified by Bradford assay (Bio-Rad, Hercules, CA, USA) and equal amounts of lysates were resolved in appropriate SDS-PAGE gels, transferred onto 0.45 μm PVDF membrane (Millipore, Banglore, India) and probed with primary antibodies. HRP-conjugated secondary antibodies and Supersignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA) were used for protein band detection using GeneGnome system (Syngene, Cambridge, UK). Anti-GAPDH alone or in combination with anti-histone-H3 antibody was used as protein loading and transfer control.
For xenograft tumour lysate preparation, excised, snap frozen tumours were homogenized in above mentioned lysis buffer using Tissue Grinder, sonicated (50% amplitude for 2 min with 10 s on and 20 s off pulses) and lysates were cleared by centrifugation.
Total proteins isolated were quantified by Bradford assay (Bio-Rad, Hercules, CA, USA) and equal amounts of lysates were resolved in appropriate SDS-PAGE gels, transferred onto 0.45 μm PVDF membrane (Millipore, Banglore, India) and probed with primary antibodies. HRP-conjugated secondary antibodies and Supersignal West Pico chemiluminescent substrate (Pierce, Rockford, IL, USA) were used for protein band detection using GeneGnome system (Syngene, Cambridge, UK). Anti-GAPDH alone or in combination with anti-histone-H3 antibody was used as protein loading and transfer control.
6
Quantitative Histone Modification Analysis
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The nuclear fractions and lysates from the untreated Skov-3 and GO/CDDP-treated Skov-3 cells were resuspended into 500 μL RIPA buffer and the protein concentration was quantified using the Bradford protein assay (Pierce). Same amount of proteins was subjected to immunoprecipitation using LC3B MAb (Cell Signaling, cat. no. 3868) and Dynabeads® Protein A (Thermo Fisher, cat. 10001D). The immunocomplex associated with LC3 was separated by 12% SDS-PAGE and the bands that were apparently upregulated after GO/CDDP treatment were cut for Liquid Chromatography/Mass Spectrometry (LC/MS/MS) analysis. The LC3-immunocomplex was also subjected to SDS-PAGE and subsequent Western blot using the following primary antibodies: rabbit MAb (1:500 dilution) specific for histone H4 (D2X4V, Cell Signaling) or acetyl-histone H4(Lys16) (Cell Signaling), mouse MAb specific for histone H1 (Merck) or rabbit polyclonal Ab specific for phosphorylated histone H1 (Merck). The secondary antibodies were HRP-conjugated goat anti-rabbit (Genetex) or anti-mouse (SeraCare) IgG. The images were developed using the chemiluminescence reagent (Western Lightning® ECL Pro, PerkinElmer) and captured using the GeneGnome system (Syngene). The band intensities were quantified using Image Pro Plus 6.0.
7
Western Blot Analysis of p53 and β-Actin
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Western blot analysis was performed as previously described by Lantto et al. [13 (link)]. Cytoplasmic proteins (15 μg) and nuclear proteins (30 μg) were first separated in 12% SDS-PAGE gel and transferred onto nitrocellulose membrane. Nonspecific binding of antibodies was blocked by 5% non-fat milk powder before exposing the membrane with a monoclonal p53 and β-actin primary antibodies (DO-7, Novocastra, Hämeenlinna, Finland; A1973, Sigma, Helsinki, Finland). To enable the visualization of proteins, the membranes were exposed to a horseradish peroxidase (HRP)-conjugated secondary antibody (HAF007, R&D Systems, Abingdon, UK) and a chemiluminescence reaction was induced by HRP-substrate (SuperSignal West Pico, Thermo Scientific, IL, USA). Proteins were detected and analyzed with GeneGnome system and GeneTools programme (Syngene, Frederick, MD, USA).
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