Gadph

Manufactured by Santa Cruz Biotechnology

Sourced in United States, United Kingdom

GADPH is a critical enzyme involved in the glycolytic pathway, responsible for the oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. It plays a fundamental role in the conversion of glucose to energy within cells.

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Lab products found in correlation

34 protocols using gadph

Western Blot Analysis of Cell Signaling

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Total protein was extracted from cells using lysis buffer containing 20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1% DTT and 1% protease inhibitor cocktail (Roche, Basel, Switzerland). Equal amounts of protein extracts (40 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred on to a PVDF membrane. Membranes were blocked with 5% w/v nonfat dry milk dissolved in Tris-buffered saline plus Tween-20 (0.1% Tween-20; pH 8.3) at room temperature for 1 h, then incubated with primary antibodies at 4°C overnight. The primary antibodies used were rabbit anti-Mad2 (Abcam, Cambridge, MA, USA), rabbit anti-Skp2 and GADPH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-Rb, pRb-Ser807/811 and pRb-S780 (Cell Signaling Biotechnology, Boston, MA, USA). After washing with Tris-buffered saline plus Tween-20, membranes were incubated with horseradish peroxidase-labeled secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature. Immunobands were visualized using enhanced chemiluminescence (ECL) kit (GE Healthcare, Waukesha, WI, USA) according to the manufacturer’s instructions and exposed to X-ray films.

Huang T., Yang L., Wang G., Ding G., Peng B., Wen Y, & Wang Z. (2017). Inhibition of Skp2 sensitizes lung cancer cells to paclitaxel. OncoTargets and therapy, 10, 439-446.

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Molecular Mechanisms of ATD Treatment

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Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS), fetal bovine serum (FBS), penicillin–streptomycin–neomycin (PSN), and trypsin–EDTA were purchased from Gibco Ltd. (Grand Island, NY, USA). Primary antibodies against p53(DO-1), p53(Pab-240), CDK2, Bax, Bcl-2, Bcl-xL pro-caspase 3/8, STAT3, MVK, GADPH, and actin were obtained from Santa Cruz Biotechnology (St. Louis, MO, USA). Anti-cyclin D1, anti-c-PARP, and anti-p-STAT3 (Tyr750) were obtained from Cell Signaling Technology (Beverly, MA, USA). Alexa 488-labeled goat anti-mouse IgG antibody was from Thermo Fisher Scientific, Waltham, MA, USA. ATD was provided by Dr. Lee and synthesized from 5-bromo-2-hydroxy-3-methoxybenzaldehyde, as previously reported [23 (link)]. Tris base and all other materials were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Tseng T.H., Wang C.J., Lee Y.J., Shao Y.C., Shen C.H., Lee K.C., Tung S.Y, & Kuo H.C. (2022). Suppression of the Proliferation of Huh7 Hepatoma Cells Involving the Downregulation of Mutant p53 Protein and Inactivation of the STAT 3 Pathway with Ailanthoidol. International Journal of Molecular Sciences, 23(9), 5102.

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Western Blotting Analysis of Adipose Tissue Proteins

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eWAT was homogenized with adipose tissue protein extraction reagent (HR7812; bjbalb, Beijing, China). Equal quantities of protein were loaded and separated on 10% SDS-PAGE gels. After electrophoresis, proteins were transferred to immobilon-P polyvinylidene difluoride membranes and blocked with 5% bovine serum albumin (BSA). Then, the membranes were immunoblotted with different primary antibodies: GLUT4 (diluted 1:200), Akt (diluted 1:200), pAkt (phosphorylated at Ser473) (diluted 1:200), and Glyceraldehyde 3-phosphate dehydrogenas (GADPH, diluted 1:500; Santa Cruz, USA). After the membranes were washed, the immunoblots were incubated with a secondary antibody conjugated with horseradish peroxidase, visualized with an ECL detection system (Syngen, Cambridge, UK), and analyzed using ChemiDoc Quantity One software (Bio-Rad Laboratories, Milan, Italy). GADPH was used as a loading control for Glut4, and pAkt was normalized to Akt.

He M.Q., Wang J.Y., Wang Y., Sui J., Zhang M., Ding X., Zhao Y., Chen Z.Y., Ren X.X, & Shi B.Y. (2020). High-fat diet-induced adipose tissue expansion occurs prior to insulin resistance in C57BL/6J mice. Chronic Diseases and Translational Medicine, 6(3), 198-207.

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Embryonic Protein Profile Analysis

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Total proteins extracted from embryos were analysed on a 10% SDS-PAGE followed by western blot analysis according to the procedures described by Lin et al. [5 (link)], except that the yolk was removed and the antibodies against Rtn4a (Abk; 1 : 1000), FAK (Cell Signaling; 1 : 1000), phosphor FAK [pY397] (Thermo; 1 : 1000), cell division control protein 42 homologue (cdc42) (New East; 1 : 500), active cdc42 (New East), α-tubulin (Sigma-Aldrich; 1 : 5000), GADPH (Santa Cruz; 1 : 1000), mouse-HRP (Santa Cruz; 1 : 5000) and rabbit-HRP (Santa Cruz; 1 : 5000) were used.

Lin C.Y., He J.Y., Zeng C.W., Loo M.R., Chang W.Y., Zhang P.H, & Tsai H.J. (2017). microRNA-206 modulates an Rtn4a/Cxcr4a/Thbs3a axis in newly forming somites to maintain and stabilize the somite boundary formation of zebrafish embryos. Open Biology, 7(7), 170009.

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Western Blot Analysis of NRF2, iNOS, IkB-α, NF-kB

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Western blots were performed as described from our previous studies [60 (link)]. Specific primary antibody: anti-NRF2 (1:600, Santa Cruz Biotechnology) or anti-iNOS (1:700; Santa Cruz Biotechnology) or anti-IkB-α (1:700; Santa Cruz Biotechnology) or anti-NF-kB (1:700; Santa Cruz Biotechnology) was mixed in 1 × PBS, 5% w/v nonfat dried milk, and 0.1% Tween 20, and were incubated at 4 °C, overnight. Afterwards, blots were incubated with peroxidase-conjugated bovine antimouse IgG secondary antibody or peroxidase-conjugated goat antirabbit IgG (1:2000, Jackson Immuno Research) for 1 h at room temperature. To verify the equal amounts of protein, membranes were also incubated with the antibody against laminin (1:1000; Santa Cruz Biotechnology) and GADPH (1:1000; Santa Cruz Biotechnology). Signals were detected with enhanced chemiluminescence detection system reagent (Super-Signal West Pico Chemiluminescent Substrate, Pierce). The relative expression of the protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS software and standardized to β-actin levels. Images of blot signals were imported to analysis software (Image Quant TL, v2003).

Fusco R., Cordaro M., Siracusa R., Peritore A.F., Gugliandolo E., Genovese T., D’Amico R., Crupi R., Smeriglio A., Mandalari G., Impellizzeri D., Cuzzocrea S, & Di Paola R. (2020). Consumption of Anacardium occidentale L. (Cashew Nuts) Inhibits Oxidative Stress through Modulation of the Nrf2/HO−1 and NF-kB Pathways. Molecules, 25(19), 4426.

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Western Blot Analysis of TRPM7, MycN, and ODC

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Cell lysates were prepared in RIPA buffer [20 mM Tris–HCl, pH 7.5, 0.1% (w/v) sodium lauryl sulfate, 0.5% (w/v) sodium deoxycholate, 135 mM NaCl, 1% (v/v) Triton X-100, 10% (v/v) glycerol, 2 mM EDTA], supplemented with Complete protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA), and phosphatase inhibitors sodium fluoride (NaF) (20 mM) and sodium vanadate (Na₃VO₄) (0.27 mM). Western blot analysis was performed as previously described [28] (link). The total protein concentration was determined using the protein assay dye reagent from Bio-Rad Laboratories (Hercules, CA, USA). Cell lysates in SDS-sample buffer were boiled for 5 min and equal amounts of total protein analyzed by 10% SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting. The antibodies used in this study are: rabbit monoclonal TRPM7 (at a 1:1000 dilution from Epitomics, Inc. (Burlingame, CA, USA)), rabbit polyclonal MycN (at a 1:500 dilution), goat polyclonal ODC (at a 1:500 dilution) and mouse monoclonal GADPH (at a 1:1000 dilution) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Secondary anti-mouse (at a 1:10,000 dilution) IR-680 or IR-800 (LI-COR Biosciences, Lincoln, NB, USA). Proteins were detected using the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NB, USA) and analyzed with Licor Image Studio 2.0 acquisition and analysis software.

Lange I, & Koomoa D.L. (2014). MycN promotes TRPM7 expression and cell migration in neuroblastoma through a process that involves polyamines. FEBS Open Bio, 4, 966-975.

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MALAT1 Knockdown Protein Expression

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The MALAT1 knockdown cells were washed twice with ice-cold PBS and treated with lysis buffer (Solarbio, China) or nuclear and cytoplasmic extraction reagent kit (Beyotime, China). Heat-denatured protein samples (25 μg per lane) were resolved by SDS poly-acrylamide gel electrophoresis (PAGE) and transferred to an Immobilon-P membrane (Millipore, Bedford, MA). The membrane was incubated with primary anti-body overnight at 4 °C, 1 h with a secondary antibody at room temperature, followed by ECL reagent (Millipore, Bedford, MA) for chemiluminescent detection. Primary antibody of MMP-2/9, TIMP-3, VEGF, Twist-1, GADPH (Santa Cruz, CA), E/N-cadherin, Zeb-1, Vimentin, Slug, β-catenin/-p and NF-κBp65 (Abcam, UK) were used for WB analysis.

Zhou X., Liu S., Cai G., Kong L., Zhang T., Ren Y., Wu Y., Mei M., Zhang L, & Wang X. (2015). Long Non Coding RNA MALAT1 Promotes Tumor Growth and Metastasis by inducing Epithelial-Mesenchymal Transition in Oral Squamous Cell Carcinoma. Scientific Reports, 5, 15972.

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Extraction and Detection of Membrane Transporters

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Membrane proteins were isolated using a protein extraction kit (Abcam, MA). Primary antibodies against human NBCe1 (anti-SlC4A4 rabbit polyclonal antibody, diluted 1:500) or NBCn1 (anti-SlC4A7 rabbit polyclonal antibody, diluted 1:500) were obtained from Abcam (Abcam, MA). The secondary antibody (anti-rabbit, diluted 1:1000) was obtained from Invitrogen (Invitrogen, MD). Anti-SlC4A4 antibody reacts to conserved amino acids at N-terminal domain of SLC4A4 and it covers all isoforms of SlC4A4. Similarly, anti-SlC4A7 antibody reacts with all the isoforms of SlC4A7. Western blotting was performed as described elsewhere. Briefly, 20 µg of lysate supernatant was separated by 4–12% SDS-PAGE and transferred to a nitrocellulose membrane. The blots were developed using ECL reagents (Bio-Rad, CA) and the ChemiDoc XRS+ molecular imager from Bio-Rad. GADPH (Santa Cruz Biotechnology, CA) was used as the loading control for all blots.

Yao H., Azad P., Zhao H.W., Wang J., Poulsen O., Freitas B.C., Muotri A.R, & Haddad G.G. (2016). The Na+/HCO3− co-transporter is protective during ischemia in astrocytes. Neuroscience, 339, 329-337.

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Crotonoside Inhibits Cell Cycle Proteins

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Six-well plates were seeded with KG-1 or MV-4-11 cells at 5 × 10⁵ cells per well. Cells were first incubated with different concentrations of crotonoside (0, 5, 10, 5, and 20 μM) for 48 h, and a 0.1% DMSO solution was used as the control vehicle. The cells were harvested and lysed using RIPA lysis buffer. The protein concentrations were confirmed by a BCA Protein Assay Kit (Beyotime) (Shanghai, China). Protein samples were fractionated using 10% polyacrylamide gels and transferred onto PVDF membranes, then blocked by 5% nonfat milk for 1 h. Blots were incubated with primary antibodies against APOBEC3G (Abcam, Cambridge, UK), CyclinB1, CDK2, CDK6, CyclinD1, and c-Myc (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C, followed by incubation with appropriate peroxidase-conjugated secondary antibodies. β-actin (Cell Signaling Technology) or GADPH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) served as an internal control. The immunocomplexes were visualized by using an enhanced chemiluminescence detection system (Millipore, Burlington, MA, USA), followed by exposing the blots to X-ray film.

Ma C., Liu P., Cui S., Gao C., Tan X., Liu Z, & Xu R. (2022). The Identification of APOBEC3G as a Potential Prognostic Biomarker in Acute Myeloid Leukemia and a Possible Drug Target for Crotonoside. Molecules, 27(18), 5804.

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Immunoblot Analysis of Protein Expression

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Immunoblot analysis (IB) was performed as previously described[24 (link)]. In brief, 50 µg of total protein was used for all IB data. Antibodies of pAkt, Akt, p GSK3β, GSK3β, β-catenin, β-actin, p Foxo3A, Foxo3A, pro-caspase 3, cleaved-caspase 3, and Bcl-XL were purchased from Cell Signaling Technology and the dilution factor was 1:1000. α-tubulin antibody was purchased from Sigma and the dilution factor was 1:3000. Antibodies of SGK1, cyclin D1, Sirt1, lamin A/C, and GADPH were purchased from Santa Cruz Biotechnology and the dilution factor was 1:500. Relative expression density was quantified using Quantity One software (Bio-Rad).

Huang C.K., Yu T., de la Monte S.M., Wands J.R., Derdak Z, & Kim M. (2015). Restoration of Wnt/β-catenin signaling attenuates alcoholic liver disease progression in a rat model. Journal of hepatology, 63(1), 191-198.

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