Colorectal tissue was lysed with 10 mM Tris–HCl (pH 7.5), 100 mM NaCl, 1% NP-40, 50 mM NaF, 2 mM EDTA (pH 8.0), 1 mM PMSF, 10 μg/ml leupeptin, and 10 μg/mL aprotinin. Equal amounts of lysate protein samples were run on SDS polyacrylamide gel and then transferred onto a PVDF membrane. Nonspecific binding was blocked by soaking the membrane in Tris buffered saline-Tween (TBST) buffer that contained 5% nonfat dry milk for 1 h. The membrane was probed overnight with an antibody against cyclooxygenase-2 (COX-2; Abcam, Cambridge, UK) and GAPDH (Cell Signaling, Danvers, MA, USA). After washing with TBST buffer, the membrane was incubated for 1 h with horseradish peroxidase-conjugated goat anti-rabbit IgG (Cell signaling). The protein levels of COX-2 and GAPDH were determined by using an enhanced chemiluminescence kit (GE Healthcare, Waukesha, WI, USA).
Gapdh
Manufactured by GE Healthcare
Sourced in United States
GAPDH is a core enzyme involved in the glycolytic pathway. It catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate, an essential step in cellular energy production.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Gapdh, by Santa Cruz Biotechnology (4 mentions)
Gapdh, by Cell Signaling Technology (3 mentions)
Pierce ecl, by Thermo Fisher Scientific (3 mentions)
Alkbh5, by Proteintech (2 mentions)
Smad7, by Santa Cruz Biotechnology (2 mentions)
Mettl3, by Abcam (2 mentions)
Mettl14, by Abcam (2 mentions)
Imagequant tl 8.2 image analysis software, by GE Healthcare (2 mentions)
Ripa buffer, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Cox 2, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by GE Healthcare (1 mentions)
Hrp conjugated anti rabbit igg, by GE Healthcare (1 mentions)
Tau13, by BioLegend (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Merck Group (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
19 protocols using gapdh
1
Colorectal COX-2 Western Blot Analysis
2
Western Blot Analysis of Protein Expression
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We lysed total protein from treated cells using RIPA buffer (Cell Signaling Technology) supplemented with Protease Inhibitor Cocktail (Thermo Fisher; 78430). Western blotting was performed as previously described41 (link), and the primary antibodies included GAPDH (Santa Cruz, sc-137179), LKB1 (Santa Cruz, sc-32245), ALKBH5 (Proteintech, 16837-1-AP), CTCF (Santa Cruz, sc-271474), METTL3 (Abcam, ab195352), METTL14 (Abcam, ab220030), FTO (Abcam, ab126605), WTAP (Proteintech, 60188-1-Ig), SOX2 (Cell signaling, #3579), SMAD7 (Santa Cruz, sc-11392), and MYC (Cell signaling, #13987). We performed densitometric analyses of band intensity using ImageQuant TL 8.2 image analysis software (GE Healthcare Life Sciences, USA) and GAPDH was as an internal control.
3
Musashi Protein Oligomerization in Alzheimer's Disease
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Western blot analyses were performed with both recombinant Musashi proteins and homogenates from AD and age-matched control brain tissues. Approximately 4 μg of Musashi oligomeric preparations and 10 μg of each brain homogenate were loaded on precast NuPAGE 4–12% Bis-Tris gels (Invitrogen) for SDS-PAGE analyses. Gels were subsequently transferred onto nitrocellulose membranes and blocked overnight at 4 °C with 10% nonfat dry milk. Membranes were then probed for 1 h at room temperature with α-Oligomeric antibody F11G3 (1:1000), α-MSI1 (1:1000, Abcam), α-MSI2 (1:1000, Abcam) and Tau13 (1:10,000, BioLegend), GAPDH (1:1000, Sigma) antibodies diluted in 5% nonfat dry milk. α-Oligomeric antibody F11G3, α- MSI1 and α- MSI2 immunoreactivity were detected with an HRP-conjugated anti-rabbit IgG (1:6000, GE Healthcare). Tau13 and GAPDH immunoreactivity were detected using an anti-mouse IgG (1:6000, GE Healthcare) diluted in 5% milk. ECL plus (GE Healthcare) was used to visualize the bands.
4
Western Blot Analysis of Ischemic Stroke
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For Western blot analysis, protein samples were obtained from the ipsilateral brain hemisphere at 1 day after tMCAO challenge. Ipsilateral brain hemisphere was triturated with neuronal protein extraction reagent containing phosphatase and protease inhibitors to extract proteins. Proteins (30 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. The membrane was blocked with 5% skim milk and incubated with primary antibodies against rabbit p-Akt, Akt, p-ERK1/2, ERK1/2, p-p38, p38, p-JNK, JNK (1:1000; cell signaling, Danvers, MA, USA), or mouse GAPDH (1:5000; Santa-Cruz Biotechnology Inc.) overnight at 4°C followed by incubation with respective secondary antibodies (1:10000; Jackson ImmunoResearch) at room temperature for 2 h. Signals were visualized with enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Rockford, IL, USA). Band intensity of each protein blot was analyzed using ImageQuantTM TL software (GE Healthcare Bio-Science, Uppsala, Sweden), normalized with GAPDH, and expressed as fold change compared to sham-operated group.
5
Oligomeric State Analysis of CysE and CysE/CysK
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The oligomeric state of CysE and CysE/CysK complex in native conditions was determined on an analytical HPLC-SEC Superdex 200 increase 3.2/300 column (GE-Healthcare, Chicago, IL, US) in PBS in the presence of 1 mM tris(2-carboxyethyl)phosphine (TCEP). A calibration curve was obtained by running five commercial standards for size exclusion chromatography (blue dextran, ferritin 440 kDa, conalbumin 75 kDa, ovalbumin 43 kDa, and carbonic anidrase 29 kDa, GE Healthcare) and the home-made standard glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 144.2 kDa).
6
Analyzing Epithelial-Mesenchymal Transition Markers
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Cells were lysed in RIPA buffer and analyzed by Western blotting as described (Huang et al. 2014 ). Antibodies used for Western blotting were as follows: anti-CD44 (R&D Systems), anti-hnRNPF (Santa Cruz Biotechnology), anti-E-cadherin (Cell Signaling Technology), anti-γ-catenin (Cell Signaling Technology), and anti-vimentin (NeoMarkers). GAPDH (GE) was used as a loading control. For immunofluorescence, cells were fixed in 4% polyformaldehyde and permeabilized with 0.2% Triton X-100 before blocking with 5% BSA. Primary antibody incubations with rabbit anti-E-cadherin (1:200; Cell Signaling Technology) were performed overnight at 4°C followed by incubation with Alexa fluor 488-conjugated anti-rabbit IgG (1:500; Invitrogen) for 2 h at room temperature and DAPI staining. Representative images were captured under a Nikon C2 laser scanning confocal microscope.
7
Western Blot Analysis of Protein Targets
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Cell lysates were prepared by using RIPA buffer. Lysates were run on an SDS-PAGE (Thermo Fisher Scientific) gel and transferred to a PVDF membrane according to the manufacturer’s protocols (Bio-Rad) at 100 V for 1 h. After blocking incubation with 5% non-fat milk in TBS-T (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween-20) for 1 h at room temperature, membranes were incubated with antibodies against GAPDH (Thermo Fisher Scientific, AM4300, 1:10,000), AID (MBL, M3-214–3, 1:2000), OsTIR1 (MBL, PD048, 1:1000), or CTCF (Santa Cruz, sc-271514, 1:200) at 4 °C overnight with gentle shaking. Membranes were washed three times for 10 min each with TBS-T and incubated with a 1:2000 (CTCF), 1:5000 (AID), or 1:20,000 (GAPDH) dilution of sheep anti-mouse IgG HRP (GE Healthcare, NA931) or 1:5000 (OsTIR1) dilution of donkey anti-rabbit IgG HRP (GE Healthcare, NA340) for 1 h at room temperature. Blots were washed with TBS-T three times for 10 min each and developed with the ECL system (Perkin Elmer) according to the manufacturer’s protocol. Uncropped raw data were provided in Additional file 4 .
8
Quantifying VEGF Expression in Periapical Lesions
9
Immunoblotting and Immunoprecipitation Protocols
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Antibodies to GAPDH and TNFR1 (TNF-α receptor 1) were purchased from R&D Systems (Minneapolis, MN, USA). Secondary antibodies (Cy2- and Cy3-conjugated) were purchased from Jackson Immunoresearch (West Grove, PA, USA). GAPDH and secondary antibodies for immunoblotting were obtained from GE Healthcare (Chicago, IL, USA). The antibody to phosphoserine (p-Serine) was bought from Abcam Company (Cambridge, MA, USA). The phospho-SRC pTyr418 antibody was purchased from Thermo Fisher Scientific Company (Waltham, MA, USA). Mounting solutions for mounting coverslips were bought from Thomas Scientific (Swedesboro, NJ, USA) or Vector Labs (Burlingame, CA, USA). The exoenzyme C3 transferase enzyme and Rho-GTP binding protein-coupled to GST beads were bought from Cytoskeleton, Inc. (Denver, CO, USA). TRAF-6 siRNA and antibodies to L-plastin, Src, TRAF-6, and GAPDH were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Polyacrylamide gel electrophoresis and protein estimation reagents were bought from Bio-Rad (Hercules, CA, USA). Rhodamine-phalloidin, Protein A-Sepharose, GST-Sepharose 4B beads, inhibitors to PKC (Gö 6983 and Staurosporine), PKA (H-89), and integrin αv (cyclo RGD peptide aka cRGDfV), and all other chemicals were purchased from Sigma (St. Louis, MO, USA).
10
SDS-PAGE Protein Detection via Western Blot
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Whole cell lysates or RNA pull-down samples were separated by 10% SDS-PAGE, transferred to a PVDF membrane (BioRad), and probed with the appropriate antibody. Primary antibodies used in western blots included HA-HRP (Roche Applied Science), hnRNPM (OriGene). GAPDH (GE) and β-actin (Sigma-Aldrich) were used as loading controls. After incubation with HRP-tagged secondary antibodies, if appropriate, blots were visualized via chemiluminescence (Thermo Fisher).
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