Gene expression levels were assessed by real-time qRT-PCR. RNA Isolation and cDNA synthesis was performed as described above. Real time qRT-PCR was performed in duplicate with 200 ng of template cDNA on an ABI Prism 7000 Sequence Detection System, using probes specific for CD44v6 (exon span V5–V6, Hs.PT.58.45400024) and total CD44 (exon span V2–V3, Hs.PT.58.4880087) (both from iDT, Corallvile, IA, USA). The relative expression level of CD44v6 and total CD44 was determined by the comparative 2−ΔΔCT method using the housekeeping gene GADPH (4352934E; ThermoFisher Scientific, Waltham, MA, USA), to normalize expression results between samples.
Gadph
Manufactured by Thermo Fisher Scientific
Sourced in United States
GADPH (Glyceraldehyde 3-phosphate dehydrogenase) is a core enzyme involved in the glycolytic pathway. It catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, a key step in cellular energy production.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Sds polyacrylamide gel, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Bcl 2, by Abcam (1 mentions)
Bcl2 associated x (bax), by Abcam (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Hsp70, by Abcam (1 mentions)
Caspase 3, by Bioss Antibodies (1 mentions)
Parp1, by ABclonal (1 mentions)
α sma, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Fibronectin, by Santa Cruz Biotechnology (1 mentions)
Col1a1, by Santa Cruz Biotechnology (1 mentions)
Las 1000 plus luminescent image analyzer, by GE Healthcare (1 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (1 mentions)
14 abi prism 7700 sequence detector system, by Thermo Fisher Scientific (1 mentions)
Dissociation curve, by Thermo Fisher Scientific (1 mentions)
Rab2a, by Thermo Fisher Scientific (1 mentions)
Fibroblast growth factor 2 (fgf2), by Merck Group (1 mentions)
7 protocols using gadph
1
CD44 Isoform Expression Analysis
2
Western Blot Analysis of Apoptosis Markers
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The cells were collected and lysed in lysis buffer (Pierce RIPA, Thermo Scientific, MA) for 20 min. After brief sonication, the lysates were centrifuged at 16,000 g for 30 min at 4 °C, and the protein content in the supernatant was measured using a Pierce BCA Protein Assay kit (Thermo Scientific). After mixing with 4 fold SDS-loading buffer (Life Technologies, Carlsbad, CA), the protein lysates were denatured at 96°C for 5 minutes, applied on an SDS polyacrylamide gel (Invitrogen, Life Technologies, Carlsbad, CA) for electrophoresis, and transferred to nitrocellulose membranes (Amersham Biosciences, Buckinghamshire, UK). Western blot analysis was performed using primary antibodies (1:1000) to caspase-3 (1:1000, Cell Signaling Technology), Bcl-2 (1:1000, Abcam), Bax (1:1000, Abcam), HSP70 (1:1000, Abcam), GADPH (1:2000, Thermofisher Scientific). The secondary horseradish peroxidase (HRP)-conjugated antibodies (1:2000) were purchased from Cell Signaling Technology. The level of protein expression was quantified by measuring the density of the immunoreactive bands and subsequently normalizing to GADPH.
3
Protein Expression Analysis in Pancreatic Tissue
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The tissue proteins extracts were harvested from fresh pancreatic tissue using RIPA lysis buffer with protease and phosphatase inhibitors (cat. nos. G2002, G2006 and G2007, Wuhan Servicebio Technology Co., Ltd.). The protein concentration was quantified using the BCA method. Total proteins (30 µg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. The PVDF membranes were blocked at room temperature for 20 min using a rapid sealing solution. The primary antibodies were used at 4°C overnight, and the antibody included Adropin (1:1,000, cat. no. PA5-72781, Thermo Fisher Scientific, Inc.); GADPH (1:10,000; cat. no. AC001, ABclonal); peroxisome proliferator-activated receptor γ (PPARγ; 1:1,000, cat. no. bsm-52220R, BIOSS); p-PPARγ Ser112 (1:1,000, cat. no. bs-3737R, BIOSS); p-PPARγ Ser273 (1:1,000, cat. no. bs-2875R, BIOSS), caspase-3 (1:1,000, cat. no. YC0006, ImmunoWay Biotechnology Company) and PARP1 (1:1,000, cat. no. A0942, ABclonal). Visualization reagent (PL101, Shenzhen SunView technology Co., Ltd.) was used, and all blotted bands were analyzed using ImageJ 1.48 software (National Institutes of Health), and the intensity values were normalized to GADPH.
4
Western Blot Analysis of Fibrotic Markers
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Protein samples were extracted from the BMFs or fBMFs. After denaturation, equal amounts of extracted proteins (30 μg) were separated by 10% SDS-PAGE with Tris-Glycine SDS running buffer and transferred with transfer buffer onto a nitrocellulose membrane (GE 446 Healthcare, Little Chalfont, Buckinghamshire, UK). Then, transferred membranes were incubated with BlockPRO blocking buffer (visual protein, energenesis biomedical co. ltd). After washing with TBST buffer (20 mM Tris, 150 mM NaCl and 0.1% Tween 20; pH 7.4) for 5 min 3 times, the transferred membranes were incubated with the primary anti-bodies against α-SMA (1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), COL1A1(1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), fibronectin (1:1000 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or GADPH (1:5000 dilution; Thermo Fisher Scientific, Waltham, MA, USA). Then, transferred membranes were washed with TBST buffer for 5 min 3 times followed by incubation with the corresponding secondary antibodies. Developed chemiluminescence signals from catalyzed ECL substrate (Pierce Biotechnology) were detected using the LAS-1000 plus Luminescent Image Analyzer (GE Healthcare, Piscataway, NJ, USA). After the intensity of each band was measured by densitometry, the relative intensities were calculated by normalizing to GAPDH.
5
Quantitative Gene Expression Analysis
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Gene expression was analyzed using TaqMan Gene expression assay (Applied Biosystems). Real-time PCR was performed on the 14 ABI/Prism 7700 Sequence Detector System (PerkinElmer/Applied Biosystems), using a pre-PCR step of 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 60°C. Specificity of the amplified products was confirmed by melting curve analysis (Dissociation Curve TM; PerkinElmer/Applied Biosystems). Samples were amplified with primers for each gene (GADPH, Hs99999905_m1; RAB2A, Hs00234094_m1; RAB2B, Hs00375685_m1; Thermo Fisher Scientific). The Ct values were normalized to the GAPDH curve. Results were quantified using the 2−ΔΔCT method. PCR experiments were performed in triplicate.
6
Evaluating LPS Response in hCMEC/D3 Cells
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The human brain microvascular endothelial cell line (hCMEC/D3) was bought from Merck and cultured with EndoGROTM-MV Complete Media Kit (Merck) supplemented with 1 ng/mL FGF-2 (Merck) in collagen-coated support. The effect of LPS stimulation on hCMEC/D3 cell line was evaluated as performed for monocytes, adding LPS (1 μg/mL) (stimulated condition) or PBS (unstimulated control condition) to the media for up to 24 h, followed by qRT-PCR as described for monocytes using GADPH (Thermo Fisher assay code: Hs02758991_g1) as endogenous control. ICC was performed as described previously. Each experiment was performed in duplicate, analyzing a total of 12 cells for each condition.
7
Quantitative RT-PCR Analysis of Steroid-Deprived MCF-7 Cells
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Steroid-deprived MCF-7 cells were stimulated with 10 mM ligands for 1 hr (MYC) or 24 hr (GREB1) in 384-well format using a Biomek NXP workstation (Beckman Coulter). Total RNA was extracted using the RNAGEM Tissue Plus RNA extraction kit (ZyGEM), reverse-transcribed using the Applied Biosystems (ABI) High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific), and analyzed in an ABI 7900HT Real-Time PCR System, using the ABI TaqMan gene expression master mix and assays for GREB1 (Hs00536412_m1), MYC (Hs00153408_m1), and GADPH endogenous control (product no. 4333764F) (Thermo Fisher Scientific).
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