Protein levels of MMP2, MMP9 and E-cadherin were detected by western blotting 24 h following treatment. In brief, the total cell proteins were extracted from cells using lysis buffer (Cell Signaling Technology Inc., Danvers, MA, USA). BCA assay (Thermo Fisher Scientific, Inc.) was carried out to measure the protein concentrations. Equal amounts of protein samples (25 µg) were separated by 12% SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. Following this, the membranes were blocked with 5% skim milk at room temperature for 2 h. Subsequently, incubation occurred with the primary antibodies against MMP2 (cat. no. 13132), MMP9 (cat. no. 2270) or E-cadherin (cat. no. 3195; all 1:1,000; all Cell Signaling Technology Inc.) overnight at 4°C. The membrane was then incubated with secondary antibodies (HRP-conjugated goat Anti-Rabbit, cat. no. Ab203-01, Vazyme, Piscataway, NJ, USA) at room temperature for 2 h. GAPDH (1:5,000; cat. no. 8884, Cell Signaling Technology Inc.) served as the internal control. Finally, to visualize the protein bands, an enhanced chemiluminescence detection system (Super Signal West Dura Extended Duration Substrate; Thermo Fisher Scientific, Inc.) was used.
Gapdh
Manufactured by Vazyme
Sourced in United States, China
GAPDH is a protein that plays a key role in the glycolytic pathway, catalyzing the conversion of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate. It is commonly used as a reference gene or loading control in various molecular biology and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin, by Cell Signaling Technology (2 mentions)
β actin, by Vazyme (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
Lysis buffer, by Cell Signaling Technology (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Vimentin, by Merck Group (1 mentions)
E cadherin, by Merck Group (1 mentions)
β catenin, by Merck Group (1 mentions)
Snail, by Merck Group (1 mentions)
Cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
Quickblock blocking buffer, by Beyotime (1 mentions)
β trcp, by Abcam (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Gapdh, by Abcam (1 mentions)
6 protocols using gapdh
1
Quantifying Protein Levels in Cells
2
Quantification of MFAP2 Expression
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Total RNA was extracted from cell samples using TRIzol reagent in accordance with the instructions of the manufacturer (Invitrogen, Carlsbad, CA, USA). We used the cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) to synthesize first-strand cDNA. Moreover, RT-PCR was carried out with the use of SYBR Green PCR Master Mix (Applied Biosystems) to detect the transcript abundance of MFAP2. The following primer pairs were used for MFAP2:
The calculation of the relative quantification was performed according to the method of 2−ΔΔCt.
Western blot analysis. For the detection of the protein expression, Western blot analysis was carried out as previously described. The antibodies used were: MFAP2 (Abcam, UK), E-cadherin (Sigma-Aldrich, Chicago, IL, USA), vimentin (Sigma-Aldrich, Chicago, IL, USA), Snail (Sigma-Aldrich, Chicago, IL, USA), β-catenin (Sigma-Aldrich, Chicago, IL, USA), and GAPDH (Vazyme, Piscataway, NJ, USA), using standard chemical luminescence methodology.
The calculation of the relative quantification was performed according to the method of 2−ΔΔCt.
Western blot analysis. For the detection of the protein expression, Western blot analysis was carried out as previously described. The antibodies used were: MFAP2 (Abcam, UK), E-cadherin (Sigma-Aldrich, Chicago, IL, USA), vimentin (Sigma-Aldrich, Chicago, IL, USA), Snail (Sigma-Aldrich, Chicago, IL, USA), β-catenin (Sigma-Aldrich, Chicago, IL, USA), and GAPDH (Vazyme, Piscataway, NJ, USA), using standard chemical luminescence methodology.
3
Protein Expression Profiling in HTR-8/SVneo Cells
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Total protein was extracted from HTR-8/SVneo cell lines using RIPA lysis buffer (Beyotime, Jiangsu, China), and the protein concentration in each extract was determined by using a BCA Kit from the same manufacturer, adhering to the protocol supplied. For each sample, 20 µg of protein was resolved via 10% SDS-PAGE and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Beyotime, Jiangsu, China). The membranes were then subsequently blocked using QuickBlock™ Blocking Buffer for (Beyotime, Jiangsu, China)1 h at room temperature. Following this step, the membranes were incubated at 4 °C overnight with primary antibodies against N-cadherin, E-cadherin (from Cell Signaling Technology, Inc, Boston, USA), as well as vimentin, snail, β-TrCP, β-actin (Abcam, Cambridge, USA), and GAPDH (Abcam, Cambridge, USA). The next stage involved incubation with an HRP-labeled secondary antibody for 1 h at room temperature. The protein bands were visualized using ECL-plus reagents (Vazyme Biotech Co., Ltd, Jiangsu, China) with GAPDH or β-actin serving as an internal control.
4
Quantitative Protein Expression Analysis
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Soluble proteins from treated cells were harvested and lysed in solution containing 50 mM HEPES (pH 7), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, 10% glycerol, 1% Triton X-100, 1 mM Na3VO4, 10 μM pepstatin, 10 μg/mL aprotinin, 5 mM iodoacetic acid, and 2 μg/mL leupeptin. Equal amounts of protein were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Roche, Basel, Switzerland). The membranes were then probed with the following primary antibodies: CD133 (#64326S, Cell Signaling Technology, Danvers, MA, USA), SOX2 (#23064 S, Cell Signaling Technology), β-catenin (#8480S, Cell Signaling Technology), Ser675-phosphorylated β-catenin (#4176S, Cell Signaling Technology), Ser9-phosphorylated GSK3β (#9336S, Cell Signaling Technology), GSK3β (#5676S, Cell Signaling Technology), PKA (#5842S, Cell Signaling Technology), GAPDH (#Ab103, Vazyme, Nanjing, China), and β-actin (#Ab101-03, Vazyme). Immunoreactivity was visualized by probing with the HRP-conjugated secondary antibody (#7074, Cell Signaling Technology) and detected using the ECL kit (#sc-2048, Santa Cruz, Santa Cruz, CA, USA). Western blot band intensities were quantified through densitometric analysis using ImageJ and normalized the band intensities with the loading control band. Original uncropped WB are given in Supplementary Material.
5
Western Blot Analysis of Cellular Proteins
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Western blot analysis was performed as standard procedures previously described [41 (link)]. The following primary antibodies directed against the antigens were used: CDH1 (1:1000, mouse monoclonal, Cell Signaling, Danvers, MA, USA), fibrillarin (1:2000, rabbit monoclonal, Cell Signaling), β-catenin (1:2000, rabbit monoclonal, Cell Signaling), Notch-1 (1:1000, rabbit polyclonal, Santa Cruz Biotechnology), p-Akt-T308 (1:1000, rabbit monoclonal, Cell Signaling), total-Akt (1:2000, mouse monoclonal, Cell Signaling), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:5000, Vazyme). GAPDH was used as the loading control in the Western blots.
6
Western Blot Analysis of AEG-1 Expression
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Western blot is widely used in biochemistry and molecular biology [39 (link), 40 (link)]. In this study, Western blot was used in detecting the expression of AEG-1 protein in different cell lines and different groups. The NSCLC cells were washed with PBS in a 6-well-plate and were lysed in a buffer of 10% glycerol, 5% SDS, 5% 2-mercaptoethanol, 0.2% romophenol blue, 80 mM Tris-HCl (ph 6.8), 5 mM EDTA (ph 8) and 1 mM phenylmethylsulfonyl fluoride. Then, the lysates were centrifuged at 12,000×g for 10 min at 4°C and were boiled for 5 min. Hybond ECL nitrocellulose membranes (GE Healthcare Bio-sciences/Amersham, Diegem, Belgium) were applied to separate the proteins for 2 h at 100 mA. The immunoblot analyses were executed using the following antibodies: AEG-1 (Cell Signaling Technology) and GAPDH (Vazyme Biotech Co., Ltd). The image pro plus 6.0 was used to analyze the IOD value.
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