Cells with a concentration of 2×104 cells/mL were cultured for 1, 4 and 7 days. Then, Trizol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) was used to isolate total RNA. After that, the extracted RNA were reversely transcribed to cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA). Purified gene specific primers for collagen type 1 (COL), osteopontin (OPN), osteocalcin (OCN), vinculin, and the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were obtained from Sangon Biotech Company (Shanghai, China), with the primer sets listed in Table 1 . Amplification was conducted as mentioned in a previous article [28 (link)].
Glyceraldehyde 3 phosphate dehydrogenase gapdh
Manufactured by Sangon
Sourced in China
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an enzyme that catalyzes the sixth step of glycolysis, the metabolic pathway that converts glucose into energy. GAPDH is responsible for the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate.
Automatically generated - may contain errors
Lab products found in correlation
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Hoxa1, by Abcam (1 mentions)
Cell counting kit 8 (cck8), by Keygen Biotech (1 mentions)
β actin, by Sangon (1 mentions)
Primescripttm rt reagent kit, by Takara Bio (1 mentions)
Rnaiso plus reagent, by Takara Bio (1 mentions)
Minocycline hydrochloride, by Merck Group (1 mentions)
Trizol reagent, by Tiangen Biotech (1 mentions)
Sybr green pcr master mix, by Takara Bio (1 mentions)
Trizol, by Takara Bio (1 mentions)
Reverse transcription kit, by Toyobo (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Lyve 1, by Santa Cruz Biotechnology (1 mentions)
P pi3k, by Immunoway (1 mentions)
Vegfr3, by Santa Cruz Biotechnology (1 mentions)
Pparα, by Sangon (1 mentions)
Caspase 3, by Sangon (1 mentions)
7 protocols using glyceraldehyde 3 phosphate dehydrogenase gapdh
1
Gene Expression Analysis of Osteogenic Markers
2
Allicin Modulates circEIF4G2 in Apoptosis
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Main reagents: Allicin (95% purity) from Nanjing Zelang Medical Technology Co., Ltd. We prepared different allicin working solutions at 20, 40, and 80 μM concentrations by using the DMEM/F‐12 medium. Furthermore, cell apoptosis, cell counting kit‐8, and two‐step reverse transcription polymerase chain reaction assay kits purchased from Nanjing KeyGEN BioTech Co., Ltd., Beyotime Institute of Biotechnology in Jiangsu and Beijing Tiangen BioTech Co., Ltd., respectively. Primers and internal reference (glyceraldehyde 3‐phosphate dehydrogenase [GAPDH]) were synthesized from Sangon Biotech Co., Ltd., Shanghai. HOXA1, AKT, and primary anti‐mTOR antibodies were all purchased from Abcam, and si‐negative control (NC) and si‐circEIF4G2 were designed and synthesized from Nanjing KeyGEN BioTech Co., Ltd.
3
Real-time PCR Analysis of 6-methoxyflavone in HeLa Cells
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Real-time fluorescence quantitative polymerase chain reaction (PCR)
Single-cell suspensions were seeded into 6-well plates for 24 h. HeLa cells were then treated with either 6-methoxyflavone (65 lM) or 0.16% DMSO for 48 h. Total RNA was extracted from 6-methoxyflavone-treated HeLa cells using RNAiso Plus reagent (Takara, Dalian, China). The complementary DNA was synthesized by genomic DNA eraser reaction and reverse transcription reaction using the PrimeScript TM RT reagent kit (Takara). Real-time PCR was performed using the TB Green V R Premix Ex Taq TM II kit (Takara). Glyceraldehyde 3phosphate dehydrogenase (GAPDH) and b-actin (Sangon Biotech, Shanghai, China) were used as housekeeping genes.
The Livak method was used to analyze the relative quantitative real-time PCR data. The real-time PCR primer sequences are listed in Table 2.
Single-cell suspensions were seeded into 6-well plates for 24 h. HeLa cells were then treated with either 6-methoxyflavone (65 lM) or 0.16% DMSO for 48 h. Total RNA was extracted from 6-methoxyflavone-treated HeLa cells using RNAiso Plus reagent (Takara, Dalian, China). The complementary DNA was synthesized by genomic DNA eraser reaction and reverse transcription reaction using the PrimeScript TM RT reagent kit (Takara). Real-time PCR was performed using the TB Green V R Premix Ex Taq TM II kit (Takara). Glyceraldehyde 3phosphate dehydrogenase (GAPDH) and b-actin (Sangon Biotech, Shanghai, China) were used as housekeeping genes.
The Livak method was used to analyze the relative quantitative real-time PCR data. The real-time PCR primer sequences are listed in Table 2.
4
Immunomodulatory Agents for Cancer Research
5
Collagen Regulation in Carbon Tetrachloride-Induced Liver Fibrosis
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PPG (Sigma, San Francisco, CA, USA); carbon tetrachloride, zinc acetate, trichloroacetic acid and p-amino-dimethyianiline dihydrochloride (Sinapharm Chemical Reagent Co., Ltd.); laminin (LN), hyaluronic acid (HA), type III procollagen (PCIII) detection kits (Naval Medical Research Institute, Shanghai, China); TRIzol (Takara, Shiga, Japan); reverse transcription kit (Toyobo, Osaka, Japan); primers for type I, type III collagen and glyceraldehyde-3- phosphate dehydrogenase (GAPDH) (Sangon, Shanghai, China); SYBR-Green PCR Master Mix (Takara).
6
Fucoidan Inhibits Lymphangiogenesis In Vitro
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The sources and characteristics of the fucoidan used in this study have been previously reported [13 (link)]. Fucoidan was diluted in Roswell Park Memorial Institute 1640 (RPMI 1640) complete medium at a concentration of 800 μg/ml. RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Life Technologies, South America). Trypsin was obtained from Hyclone (Thermo Fisher Scientific, Carlsbad, CA, USA).
The following primary antibodies were used in this study: VEGFR3, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and phospho-Akt (p-Akt; Santa Cruz, CA, USA); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cyclin-dependent kinase 4 (CDK4; Sangon Biotech, Shanghai, China); PROX1 and cyclin D1 (Boster, Wuhan, China); NF-κB (Zhongshan Biotech, Beijing, China) and p-PI3K (ImmunoWay, Newark, DE, USA).
The following primary antibodies were used in this study: VEGFR3, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) and phospho-Akt (p-Akt; Santa Cruz, CA, USA); glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and cyclin-dependent kinase 4 (CDK4; Sangon Biotech, Shanghai, China); PROX1 and cyclin D1 (Boster, Wuhan, China); NF-κB (Zhongshan Biotech, Beijing, China) and p-PI3K (ImmunoWay, Newark, DE, USA).
7
Regulation of PPARα and Caspase-3 in PANC-1 Cells
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PANC-1 cells were treated separately with DMSO (control), 60 μg/mL EGCG, 20 μM Y-27632, and 60 μg/mL EGCG + 20 μM Y-27632 for 48 h. Quantitative real-time polymerase chain reaction (RT-qPCR) [19 (link)] was used to observe the expression of PPARα mRNA and caspase-3 mRNA of these groups. The primer pairs [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), PPARα, and caspase-3] were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The primer pairs included the following: forward: 5′-AGAAGGCTGGGGCTCATTTG-3′ and reverse: 5′-AGGGGCCATCCACAGTCTTC-3′ for GAPDH (258 bp); forward: 5′-TTCGCAATCCATCGGCGAG-3′ and reverse: 5′-CCACAGGATAAGTCACCGAGG-3′ for PPARα (146 bp). Forward: 5′-CATGGAAGCGAATCAATGGACT-3′ and reverse: 5′-CTGTACCAGACCGAGATGTCA-3′ for caspase-3 (139 bp). GAPDH was used as an internal control to evaluate the relative expression of PPARα. RT-qPCR reagents were purchased from TIANGEN Biotech (Beijing) Co., Ltd. (Beijing, China). Relative mRNA was calculated using the formula: 2−ΔΔCt [20 (link),21 (link)].
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