The expression levels of proteins in GCAFs and cancer cells were assessed as follows. Total cellular protein samples were prepared from cell lysates in lysis buffer. After the protein concentration of each sample was adjusted, sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis was carried out to separate proteins. Subsequently, the protein bands obtained were transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were incubated with specific primary antibodies against SPARC (1:1000 dilution, CST, MA, USA), PARP (1:1000 dilution, CST, MA, USA), caspase 3 (1:1000 dilution, CST, MA, USA), Bak (1:1000 dilution, CST, MA, USA), Bax (1:1000 dilution, CST, MA, USA), tubulin (1:1000, CST, MA, USA), and GAPDH (1:1000, CST, MA, USA) at 4 °C overnight. GAPDH and tubulin were used as the internal controls. The expression levels of the target proteins were assessed via an ECL detection system (Merck, Darmstadt, Germany) on a Syngene GeneGenius gel imaging system (Syngene, Cambridge, UK). And the relative changes of protein expression were analysed using Image J software.
Genegenius gel imaging system
Manufactured by Syngene
Sourced in United Kingdom, Germany
The GeneGenius Gel Imaging System is a laboratory instrument designed for the visualization and analysis of nucleic acid and protein gels, such as those used in electrophoresis experiments. The system includes a high-resolution camera, a specialized light source, and image processing software to capture and analyze gel images. The GeneGenius Gel Imaging System provides users with a reliable and efficient tool for documentation and quantification of gel-based experiments.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (6 mentions)
Ecl detection reagent, by Merck Group (4 mentions)
Ecl detection system, by Merck Group (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
P pten, by Cell Signaling Technology (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
P mtor, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
P70s6k, by Cell Signaling Technology (1 mentions)
Surepage gel, by GenScript (1 mentions)
Rabbit anti mad 1 monoclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Ab185924, by Abcam (1 mentions)
Ab37185, by Abcam (1 mentions)
Thermocycler, by Thermo Fisher Scientific (1 mentions)
Proteinase inhibitor, by Roche (1 mentions)
Mj research ptc 200 thermal cycler, by Bio-Rad (1 mentions)
16 protocols using genegenius gel imaging system
1
Quantifying Protein Expression in Cancer
2
AKT-mTOR Signaling Pathway Protein Analysis
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The expression levels of proteins in cancer cells were examined as follows. Total cellular proteins were prepared from cell lysates with lysis buffer. For AKT-mTOR signalling pathway protein detection, cells were treated with OE/VEC CM for 30 minutes before protein extraction. After the protein concentration of each sample was adjusted, SDS-polyacrylamide gel electrophoresis was performed to separate proteins. Subsequently, the protein bands were transferred to a polyvinylidene fluoride (PVDF) membrane. The specific primary antibodies were used as follows: p-PTEN (1:1000, Cell Signaling Technology, Massachusetts, USA), p-AKT (1:1000, Cell Signaling Technology, Massachusetts, USA), AKT (1:1000, Cell Signaling Technology, Massachusetts, USA), p-mTOR (1:1000, Cell Signaling Technology, Massachusetts, USA), mTOR (1:1000, Cell Signaling Technology, Massachusetts, USA), p-70S6K (1:1000, Cell Signaling Technology, Massachusetts, USA), and GAPDH (1:1000, Cell Signaling Technology, Massachusetts, USA) at 4°C overnight. GAPDH served as the internal control. The levels of target proteins were detected using the ECL detection system (Merck, Darmstadt, Germany) and the Syngene GeneGenius gel imaging system (Syngene, Cambridge, UK). The relative changes in protein expression were analysed using ImageJ software.
3
Protein Extraction and Western Blot Analysis
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Total cellular protein was collected in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing phenylmethylsulfonyl fluoride (PMSF), aprotinin, sodium orthovanadate and NaF. Equal amounts of proteins were subjected to electrophoresis on a SurePAGE gel (GenScript, Nanjing, China) and then transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked in 5% skimmed milk for 1 h at room temperature and incubated with different diluted primary antibodies, including HMGA2 (1:1000 dilution, CST, MA, USA), PARP (1:1000 dilution, CST, MA, USA), cleaved PARP (1:1000 dilution, CST, MA, USA), caspase-3 (1:1000 dilution, CST, MA, USA), cleaved caspase-3 (1:1000 dilution, CST, MA, USA), caspase-7 (1:1000 dilution, CST, MA, USA), cleaved caspase-7 (1:1000 dilution, CST, MA, USA), P-ERK (1:2000 dilution, CST, MA, USA), ERK (1:1000 dilution, CST, MA, USA), tubulin (1:1000 dilution, CST, MA, USA), and GAPDH (1:1000 dilution, CST, MA, USA), at 4 °C overnight. Finally, an enhanced chemiluminescence (ECL) detection system (Merck, Darmstadt, Germany) and the Syngene GeneGenius gel imaging system (Syngene, Cambridge, UK) were used to visualize the protein bands after incubation with secondary antibody.
4
Protein Extraction and Western Blot Analysis
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Total protein was extracted using RIPA buffer and the extracts containing equal quantities of protein (30 μg) were electrophoresed in 10% polyacrylamide gel, transferred with PVDF membranes and blocked for 1 h (5% BSA in TBS-Tween 20 buffer) at room temperature. Incubations with primary antibodies to detect CD44, CBS, and β-actin (CST, USA) were followed by incubations with secondary antibodies conjugated with horseradish peroxidase (CST, USA). Blots were developed with ECL detection reagents (Millipore, USA). Images were collected utilizing Syngene GeneGenius gel imaging system (Syngene, UK) according to the manufacturer's instructions.
5
Western Blot Analysis of Nuclear Proteins
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Total protein was extracted using the method described previously with little modifications [22] (link). Nuclear protein was extracted using NucBuster™ protein extraction kit (Novagen, German) following the manufacturer's recommendations. The concentrations of protein were determined using BCA kit (ThermoFisher, USA). Then, the extracts containing equal quantities of protein (30 μg) were electrophoresed in 6% or 10% polyacrylamide gel. Subsequently, the separated proteins were transferred onto a PVDF membrane. The membrane was then blocked for 1 h (5% bovine serum albumin (BSA) in TBS-Tween 20 buffer) at room temperature and then incubated overnight at 4°C with rabbit anti-VDR monoclonal antibody (1:1000 dilution, Abcam, USA), rabbit anti-Mad-1 monoclonal antibody (1:100 dilution, Santa Cruz), rabbit anti-C-Myc monoclonal antibody (1:1000 dilution, CST, USA), rabbit anti-Histone H3 monoclonal antibody (1:1000 dilution, Abcam, USA) and rabbit anti-GAPDH monoclonal antibody (1:1000 dilution, CST, USA). The membranes were subsequently incubated at room temperature for 1 h with corresponding secondary antibodies (1:10,000 dilution, CST, USA) and blots were developed with ECL detection reagents (Millipore, USA). Images were collected utilizing Syngene GeneGenius gel imaging system (Syngene, UK) following the manufacturer's instructions.
6
Western Blot Analysis of Protein Markers
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Proteins extracts were analyzed by western blotting following standard protocols with primary antibodies specific for xCT (ab37185, Abcam), CBS (#14782, CST), CTH (#60234-1-Ig, Proteintech), MPST (sc-376168, Santa cruz), GAPDH (#5174, CST), OTUB1 (#3783, CST), CD44s (ab185924, Abcam), PARP (#9532, CST), Cleaved PARP (#5625, CST), Caspase 3 (#9662, CST) and Cleaved Caspase 3 (#9664, CST). Images were collected utilizing Syngene GeneGenius gel imaging system (Syngene, UK) following the manufacturer's instructions.
7
Genomic DNA Extraction and SSR Genotyping
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Genomic DNA was extracted from the young leaves of the selected genotypes following CTAB (cetyltrimethyl ammonium bromide) procedure. Purified DNA was subjected to PCR amplification in 20 μl reaction mixture containing 5.0 μl DNA (20 ng/μl), 2.0 μl 10× buffer, 2.0 μl dNTPs (25 mM), 2.0 μl each forward and reverse SSR primers (30 ng/μl), 0.3 μl TaqDNA polymerase (3U/μl) and 6.7 μl double distilled water. Amplification of the template DNAs was performed in thermocycler (Applied Biosystem) as per the following profile: DNA was denatured at 94 °C for 2 min. followed by 35 cycles each consisting of denaturation at 94 °C for 1 min., primer annealing at 49 °C for 2 min., primer elongation at 72 °C for 3 min. Final elongation of the amplicons was allowed to complete at 72 °C for 10 min which was finally put on hold at 4 °C. Amplified products so obtained were resolved on 3 % metaphore agarose gel stained with ethidium bromide and analyzed in Gene Genius Gel Imaging System from Syngene.
8
Western Blot Analysis of EMT Markers
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RIPA lysis buffer (Sigma, USA) supplemented with proteinase inhibitor (Roche Applied Science, Indianapolis, IN, USA) was managed to extract total protein, whose concentration was determined via BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Exactly 30 μg protein extracts were electrophoresed within 6%‐10% polyacrylamide gel and were then transferred onto the PVDF membrane. Subsequently, the membranes were blocked at room temperature for 1 hour, and we added rabbit anti‐human monoclonal antibodies for E‐cadherin, vimentin, and N‐cadherin (1:1000, Proteintech, Manchester, UK), along with rabbit anti‐human GAPDH monoclonal antibody (1:1000, CST, Danvers, MA, USA). After that, corresponding mouse anti‐rabbit secondary antibodies for E‐cadherin, vimentin, and N‐cadherin (1:10 000, CST) were supplemented for 1‐hour culture at room temperature. Finally, ECL detection reagent (Millipore, Billerica, MA, USA) was applied for development, and images were analyzed with Gene Genius gel imaging system (Syngene, Cambridge, UK).
9
Quantitative RNA Extraction and Analysis
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The total RNA from the transfected cells was extracted using a one-step extraction method with TRIzol (Invitrogen). To assess the RNA quantity and quality, absorbance at 260 nm and 280 nm was measured using a microplate reader (Epoch, BioTek). For each sample, 500 ng of RNA was used as the template in a 10-μl cDNA transcription system. A standard reverse-transcription (RT) protocol supplied by Promega (utilizing MMLV) was used with oligo(T)18. This procedure was followed by PCR amplification and product separation on 2% agarose gels or Q-PCR amplification using an IQ5 (Bio-Rad). In all the PCR or Q-PCR systems, 1 μl of cDNA was used as the template. Gel images were captured using a GeneGenius Gel Imaging System (SynGene) and quantified with the associated GeneTools analysis software. The primer sequences can be found in Supplementary Data 2.
10
Protein Expression Analysis in Cells
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The group division was the same as that used in the cell viability and apoptosis assay. Total proteins were separated by 4-12% SurePAGE and transferred onto a PVDF membrane. After blocking in 5% BSA for 1 hour, the bands were incubated with the primary antibodies overnight at 4 °C, followed by incubation with the corresponding secondary antibodies for 1 hour. The membranes were washed with TBST after incubation with each antibody. The specific primary antibodies were used as follows: PARP (1:1000 Dilution; CST, MA, USA), cleaved PARP ( 1:1000 dilution, CST, MA, USA), P53 (1:1000 dilution, CST, MA, USA), cleaved caspase 3 (1:1000 dilution, CST, MA, USA), caspase 9 (1:1000 dilution, CST, MA, USA), Bcl-2 (1:1000 dilution, CST, MA, USA), Bax (1:1000 dilution, CST, MA, USA) and GAPDH (1:1000, CST, MA, USA). GAPDH served as the internal controls. The bands were detected using the Syngene GeneGenius gel imaging system (Syngene, Cambridge, UK). The results were analyzed by ImageJ software.
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