Total protein was extracted with RIPA buffer, and 20–50 μg samples were loaded after measuring their concentration using a BCA kit and separated by 6%, 8% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were transferred onto polyvinylidene fluoride membrane blocked with 5% fat-free milk for 2 h at room temperature and incubated in primary antibodies against ADAM8 (1:1000, Proteintech), HB-EGF (1:1000, Abclonal Technology), EGFR (1:1000, Proteintech), p-EGFR (1:1000, CST, Beverly, MA, USA), ERK (1:1000, CST), p-ERK (1:1000, CST), AKT (1:1000, Proteintech), p-AKT (1:1000, Proteintech), CCL2 (1:1000, Proteintech), and GAPDH (1:2000, Enogene, New York, NY, USA) at 4 °C overnight. Membranes were washed and incubated in horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. An enhanced chemiluminescence system (NCM Biotech, Suzhou, China) was used to detect the protein bands.
Gapdh
Manufactured by Enogene
Sourced in China, United States
GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is an enzyme that catalyzes the sixth step of glycolysis. It is responsible for the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, which is a key step in the breakdown of glucose to produce energy for the cell.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Proteintech (2 mentions)
E cadherin, by Enogene (2 mentions)
P akt, by Enogene (2 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (2 mentions)
Ly294002, by Merck Group (2 mentions)
Hb egf, by ABclonal (1 mentions)
Adam8, by Proteintech (1 mentions)
β actin, by Merck Group (1 mentions)
Bcl 2, by Boster Bio (1 mentions)
Caspase 3, by Boster Bio (1 mentions)
β actin, by Boster Bio (1 mentions)
Bcl2 associated x (bax), by Boster Bio (1 mentions)
Anti nf κb antibody, by Proteintech (1 mentions)
Stub1, by Proteintech (1 mentions)
Ferritin, by Cell Signaling Technology (1 mentions)
Bcl 2, by Enogene (1 mentions)
Bcl2 associated x (bax), by Proteintech (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using gapdh
1
Protein Extraction and Western Blot Analysis
2
Protein Expression Analysis in Cell Lysates
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Protein expression was analysed from whole cell lysates as previously described25 , using primary antibodies recognising GRP78 (BiP) (C50B12 #3177, Cell Signaling Technology (CST)), CHOP (DDIT3, ab11419, Abcam), PTEN (#9552, Cell Signaling Technology), PTP1B (N-terminal, SAB4502525, Sigma-Aldrich), insulin receptor β (4B8 #3025, Cell Signaling Technology), β-actin (#A1978 Sigma-Aldrich), and GAPDH (#E12-042, EnoGene), p-PERK (#3179, CST), total PERK (#3192, CST).
3
Molecular Mechanisms of Anti-Cancer Effect
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MTT, ethidium bromide (EB), 2,2’-di-pyridylketone, RPMI-1640, and other chemicals were purchased from Sigma-Aldrich. Fetal bovine serum was purchased from Every Green Zhejiang Tianhang Technology Co. Ltd. (Hang Zhou, China). Anti-NF-κB antibody was obtained from Proteintech Group (Wuhan, China); Antibodies against vimentin, slug, caspase 3, β-actin, Bax, and Bcl-2 were purchased from Boster (Wuhan, China). Antibodies against AKT, p-AKT, mTOR, E-cadherin, and Gapdh were purchased from Enogene (Nanjing, China). 4′-demethylpodophyllotoxin was purchased from Shanghai PureOne Biotechnology (Shanghai, China)
4
Molecular Mechanisms of Cell Death
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MTT, 3-methyladenin (3-MA), pifithrin-α (PFT-α), RPMI-1640, and other chemicals were purchased from Sigma-Aldrich. Gapdh, NCOA4, Bcl-2, and AKT antibody were obtained from EnoGene (Nanjing, China); antibodies EGFR, stub-1, SOD, MDM2, Cyt-C, LC3, Bax, and p-AKT were purchased from Proteintech Group Inc. (Wuhan, China). Antibodies p53, p-p53, and ferritin were purchased from Cell Signaling Technology (Massachusetts, USA); siRNA for MDM2 (stB0001232) and stub1 (stB0001238) were obtained from RiboBio (Guangzhou, China).
5
Molecular Mechanisms of Autophagy and Apoptosis
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RPMI-1640 medium was purchased from Gibco; Thermo Fisher Scientific (Grand Island, NY, USA). The autophagy inhibitors 3-methyladenine (3-MA) and LY294002 were obtained from Sigma-Aldrich; Merck KGaA (St. Louis, MO, USA). Rabbit anti-human/mouse/rat polyclonal antibody LC3 (E18-5402, 1:500), GAPDH (E1C604, 1:2,000), cytochrome oxidase (COX) IV (E12-327, 1:1,000), cleaved caspase-3 (E11-0104L, 1:500), cleaved caspase-8 (E18-5267, 1:1,000), cleaved caspase-9 (E18-5240, 1:1,000), mouse anti-human/mouse monoclonal antibody Bcl-2 (E10-30077, 1:1,000), rabbit anti-human/mouse polyclonal antibody Bax (E11-0773B, 1:500), Beclin-1 (E90562, 1:500), mouse anti-human/mouse/rat monoclonal antibody cytochrome c (E12-378, 1:2000), goat anti-rabbit (E0L3012), goat anti-mouse IgG secondary antibodies (E0L3032), Cell Counting Kit-8 (CCK-8) and cell apoptosis analysis kit were all purchased from EnoGene (New York, NY, USA). Mouse anti-human p53 polyclonal antibody was obtained from BD Pharmingen (554294; San Jose, CA, USA). TRIzol reagent was obtained from Life Technologies; Thermo Fisher Scientific (Carlsbad, CA, USA). RevertAid First Strand cDNA Synthesis kit and QuantiFast SYBR Green PCR kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Mitochondrial membrane potential assay kit with JC-1 was obtained from Beyotime Biotechology (Shanghai, China).
6
LoVo Colorectal Cancer Cell Protocol
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The human colorectal cancer LoVo cells were purchased from ATCC (Manassas, VA, USA). LoVo cells were cultured in F12K medium supplemented with 10% fetal bovine serum, 100 g/mL streptomycin, and 100 U/mL penicillin, at 37°C, 5% CO2, and high humidity. The sources of antibodies (Abs) were as follows: IL-6 was purchased from R&D (Minneapolis, MN, USA), p-STAT3 was purchased from Abcam (Cambridge, MA, USA), cleaved caspase-3 was purchased from Cell Signaling (Beverly, MA, USA), and STAT3, cyclin D1, GAPDH, and the HRP-labeled secondary antibodies were purchased from EnoGene (Nanjing, China). Carboplatin was purchased from MelonePharma (Dalian, Liaoning, China). Annexin-V-FITC apoptosis detection kit, DAB Substrate Kit, and Cell Counting Kit-8 (CCK-8) were purchased from Beyotime (Beyotime, Shanghai, China). IL-6 ELISA kit was from NeoBioscience (Shenzhen, Guangdong, China).
7
Epithelial-Mesenchymal Transition Regulation
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MTT, PFT-α, TGF-β1, di-2-pyridylketone, RPMI-1640, LY294002, and other chemicals were purchased from Sigma-Aldrich (Shanghai, China). Fetal bovine serum was purchased from Every Green Zhejiang Tianhang Technology Co. Ltd. (Hangzhou, China). Antibodies of vimentin, slug, snail, and p53 were purchased from Boster (Wuhan, China). Antibodies of AKT, p-AKT, mTOR, p-mTOR, E-cadherin, and Gapdh were purchased from EnoGene (Nanjing, China). 4′-Demethylpodophyllotoxin was purchased from Shanghai PureOne Biotechnology (Shanghai, China).
8
Melanogenesis Regulation in Melanocytes
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Morin was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies against MITF (#12590), p-ERK (#9101), ERK (#9102), p-JNK (#4668), p-p38 (#4511), and PD98059 were purchased from Cell Signaling Technology (Beverly, MA, USA). GAPDH was purchased from Enogene (Nanjing, China). TRP-1 (sc-166857), JNK (sc-571), and p38 (H-147) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TRP-2 (ab221144) was purchased from Abcam (Cambridge, MA, USA). DMEM, fetal bovine serum (FBS), penicillin/streptomycin, and trypsin-ethylenediaminetetraacetic acid were purchased from Gibco (Grand Island, NY, USA). Dimethyl sulfoxide (DMSO), α-MSH, NaOH, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and 3,4-dihydroxy-L-phenlyanine (L-DOPA) were obtained from Sigma-Aldrich (St. Louis, MO, USA). SP600125 and SB203580 were purchased from Cayman Chemical (Ann Arbor, MI, USA). Pro-Prep lysis buffer was purchased from iNTRON Biotechnology (Seoul, Korea).
9
Notch-1 Ubiquitination in T-Leukemia
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T-Leukemia cell lines Jurkat were cultured with 2 mM ketamine for 10 h and collected for WB. The Antibodies used were Notch-1 antibody (Cell Signaling 3608, 1:1000), GAPDH (Enogene E12-052-4, 1:5000). For Notch ubiquitin assay, 5 μM MG132 (Sigma) was added with or without ketamine into the media, Jurkat cells were then lysed and precipitated with Notch-1 antibody (1:50) and then bound with protein A/G (Santa Cruz sc-2003). Anti-ubiquitin (Santa Cruz sc-8017) antibody were applied to detect ubiquitinated proteins.
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