Ovaries from 1 dpp female mice, 2000 ng oocytes and 100 oocytes of each size were washed in PBS containing 0.1% polyvinyl alcohol and lysed in sample buffer (0.25 m Tris–HCl (pH 6.8), 40% glycerol, 0.8% SDS and 1% β-mercaptoethanol). Proteins were separated by SDS–PAGE in 5−20% gradient gels (ATTO, Tokyo, Japan) and transferred to PVDF membranes. Blocking and immunoreactions were performed with the Can Get Signal Immunoreaction Kit (TOYOBO, Osaka, Japan) and the following antibodies: anti-Dnmt3a (IMGENEX, IMG-268A, CA, USA; 1:2000), anti-Dnmt3L (gifted from Shoji Tajima, 1:2000), anti-GAPDH (Trevigen, Gaithersburg, UK, 1:2000) and anti-α-tubulin (Sigma-Aldrich, 1:2000). Western blot experiments were repeated at least three times.
Anti gapdh
Manufactured by Bio-Techne
Sourced in United States, United Kingdom
Anti-GAPDH is a laboratory reagent used to detect and quantify the presence of the GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) protein in biological samples. GAPDH is a widely used housekeeping gene and its protein is involved in the glycolytic pathway. Anti-GAPDH can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and ELISA, to study the expression levels of GAPDH in different cell types and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Anti α tubulin, by Merck Group (3 mentions)
Can get signal immunoreaction kit, by Toyobo (2 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (2 mentions)
Phospho mlc2 and mlc2 antibodies, by Cell Signaling Technology (2 mentions)
Phospho akt and total akt antibodies, by Cell Signaling Technology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions)
Anti dnmt3a, by Novus Biologicals (1 mentions)
Phospho ampkα thr172, by Cell Signaling Technology (1 mentions)
Anti β actin, by Abcam (1 mentions)
Image lab software 5, by Bio-Rad (1 mentions)
Anti ampk, by Cell Signaling Technology (1 mentions)
Anti lc3, by MBL International Corporation (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Anti ndufs4, by Abcam (1 mentions)
Lumi light pod substrate, by Roche (1 mentions)
Anti sdha, by Abcam (1 mentions)
Anti atp5a, by Proteintech (1 mentions)
Anti uqcrc2, by Merck Group (1 mentions)
Anti mtco2, by Abcam (1 mentions)
Western blocking reagent, by Roche (1 mentions)
12 protocols using anti gapdh
1
Profiling Oocyte Dnmt3a and Dnmt3L Levels
2
Galectin-3 Signaling in Liver Homeostasis
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Benign liver and tumor tissue was homogenized in RIPA buffer with protease inhibitor cocktails. The protein concentration was determined with the Bio-Rad protein assay kit (Thermo Scientific) and 20 μg of protein was separated by SDS–PAGE. The blot was probed with anti-galectin3 (kindly provided by Dr. F-T Liu, UC Davis) and anti-GAPDH (Trevigen, Gaithersburg, MD) as internal control. Hepa1-6 cells were transfected with scrambled or galectin3 siRNA, as above. Fifty μg of protein was used for Western blot. The blots were incubated with the appropriate antibodies for 16h at 4 C: anti-phospo-NFκB (p65) (Cell Signaling Techology), phospho MLC2 and MLC2 antibodies (Cell Signaling Technology), phospho-Akt and total Akt antibodies (Cell Signaling Technology) or the anti-galectin 3 antibody. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) and the blots were developed by enhanced chemiluminescence (Thermo Scientific, Rockford, IL). The intensity of signals was quantified with ImageJ (National Institutes of Health, Bethesda, MD). The data were normalized to the expression of anti-GAPDH antibody.
3
Western Blot Analysis of Autophagy and AMPK Signaling
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SK-N-BE(2) tumor and mouse muscle tissues (50-100 mg) were homogenized with an Ultra-Turrax homogenizer (IKA, Staufen, Germany) in 10-fold extraction buffer (20 mM Tris-HCl, pH 7.6, 250 mM sucrose, 40 mM KCl, 2 mM EGTA) and then homogenized with a motor-driven Teflon-glass homogenizer (Potter S, Braun, Melsungen, Germany). The homogenates were centrifuged at 600×g for 10 min at 4°C. A total of 20 µg protein was used for Western blot analysis, as previously described [95 (link)]. The following primary antibodies were diluted in TBS-T containing 10% western blocking reagent (Roche): 1:1000 anti-LC3 (MBL International Corporation, Vienna, Austria), 1:1000 Phospho-AMPKα (Thr172) (Cell Signaling Technologies (#2535), Frankfurt, Germany), 1:1000 anti-AMPK (Cell Signaling Technologies (#2532), Frankfurt, Germany), 1:5000 anti-beta Actin (Abcam, Cambridge, UK) and 1:5000 anti-GAPDH (Trevigen, Vienna, Austria). Horseradish peroxidase-labeled secondary antibodies were used (Dako, Vienna, Austria). Detection and quantification of band intensities was performed using Image Lab Software 5.2.1 (Bio-Rad) and normalized to the corresponding loading control.
4
Galectin-3 Signaling in Liver Homeostasis
5
Western Blot Analysis of Mitochondrial Proteins
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600g homogenates were used for western blot analysis as described before [3 (link)]. In brief, a total of 10 μg protein was separated on 10% acrylamide/bis-acrylamide gels and transferred to nitrocellulose membranes (Amersham Biosciences) using a CAPS buffer (10 mM 3-[cyclohexylamino]-1-propane sulfonic acid, pH 11; 10% methanol). The following primary antibodies were diluted in 1% western blocking reagent (Roche Diagnostics) dissolved in TBS-T: anti-GAPDH (Glyceraldehyde-3-phosphate dehydrogenase, 1:10000, Trevigen), anti-NDUFS4 (NADH dehydrogenase [ubiquinone] iron-sulfur protein 4; 1:2000, Abcam), anti-SDHA (Succinate dehydrogenase [ubiquinone] flavoprotein subunit; 1:5000, Abcam), anti-UQCRC2 (Cytochrome b-c1 complex subunit 2; 1:1000, Sigma), anti-MTCO2 (Cytochrome c oxidase subunit 2; 1:5000, Abcam), anti-ATP5A (ATP synthase subunit alpha; 1:1000, Protein Tech), anti-SCOT (Succinyl-CoA:3-ketoacid coenzyme A transferase 1; 1:1000, Abnova). Horseradish peroxidase-labeled secondary antibodies were used at a dilution of 1:1000 (Dako) and detection was carried out with Lumi-Light POD-substrate (Roche).
6
NSCLC Cell Culture and Characterization
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Human NSCLC cell lines, A549 and Lu99 cells, were obtained from American Type Culture Collection, Manassas, and Riken Bioresource Center, Japan, respectively. The cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) (Nichirei Biosicence Inc., Tokyo, Japan) [41 (link),51 (link)]. Dinactin of ≥95.00% purity (Cat.No. C3454) was purchased from the APExBIO (Boston, MA, USA). Antibodies for Cyclin-A, Cyclin-B, Cyclin-D3, Cdk2, and PCNA (BD Transduction Laboratories™, San Jose, CA, USA), p21 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-GAPDH (Trevigen, Minneapolis, MN, USA) were used for the experiments. Metformin was purchased from Sigma-Aldrich (Burlington, MA, USA).
7
Western Blot Quantification of HER2, Tubulin, and GAPDH
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Western blot was carried out as previously described [14 (link)]. The following antibodies were used: mouse monoclonal anti-HER2 (BioGenex, CB11), rabbit polyclonal anti-β-Tubulin (Santa Cruz, H-235) and rabbit polyclonal anti-GAPDH (Trevigen). Densitometry quantification was performed using ImageJ software.
8
Characterization of DNMT3A2 Expression
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Female 2lox mice were crossed with Vasa-Cre males, and their offspring were used as ovarian and oocyte donors. All samples
were washed in PBS and lysed in sample buffer (0.25 M Tris-HCl, pH 6.8; 40% glycerol; 0.8% SDS; and 1% β-mercaptoethanol).
SDS-PAGE was performed using 5–20% gradient gels (ATTO, Tokyo, Japan), and samples were transferred to PVDF membranes.
Blocking and immunoreactions were performed using a Can Get Signal Immunoreaction Kit (Toyobo) with the following antibodies:
anti-DsRed (1:2,000; Takara Bio, Otsu, Shiga, Japan), anti-DNMT3a (IMG-268A, 1:2,000; Imgenex, San Diego, CA, USA),
anti-GAPDH (1:2,000; Trevigen, Gaithersburg, MD, USA) and anti-αTubulin (1:2,000, Sigma-Aldrich Japan). The expression level
of DNMT3A2 was normalized using GAPDH or αTubulin expression.
were washed in PBS and lysed in sample buffer (0.25 M Tris-HCl, pH 6.8; 40% glycerol; 0.8% SDS; and 1% β-mercaptoethanol).
SDS-PAGE was performed using 5–20% gradient gels (ATTO, Tokyo, Japan), and samples were transferred to PVDF membranes.
Blocking and immunoreactions were performed using a Can Get Signal Immunoreaction Kit (Toyobo) with the following antibodies:
anti-DsRed (1:2,000; Takara Bio, Otsu, Shiga, Japan), anti-DNMT3a (IMG-268A, 1:2,000; Imgenex, San Diego, CA, USA),
anti-GAPDH (1:2,000; Trevigen, Gaithersburg, MD, USA) and anti-αTubulin (1:2,000, Sigma-Aldrich Japan). The expression level
of DNMT3A2 was normalized using GAPDH or αTubulin expression.
9
Western Blot Analysis of Cell Signaling
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Total cell lysates were processed for western blotting as previously described and probed with the following antibodies: rabbit monoclonal anti-Nox4 (UOTR1B493) (Abcam, Cambridge, MA, USA); mouse monoclonal anti-p53 (clone DO-1; Santa Cruz Biotechnology, Dallas, TX, USA); rabbit monoclonal anti-phospho-SMAD3 (clone EP823Y; Abcam); rabbit polyclonal anti-phospho-SMAD2 (no. 3101; Cell Signaling, Beverly, MA, USA); rabbit monoclonal anti-SMAD3 (clone EP568Y; Abcam); mouse monoclonal anti-SMAD2 (no. L16D3; Cell Signaling); rabbit monoclonal anti-phospho-FAK (Y576; Invitrogen); rabbit polyclonal anti-FAK (no. 3285; Cell Signaling); (rabbit polyclonal anti-GAPDH (Trevigen); and mouse monoclonal anti-V5 (Invitrogen) antibodies.
10
Western Blot Analysis of Protein Targets
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Whole protein was extracted by M-PER Mammalian Protein Extraction Reagent (Thermo) from cell lines added with Phosphatase Inhibitor Cocktail Set II (Calbiochem, San Diego, CA) and Complete Protease Inhibitor Cocktails (Roche, Lewes, UK) according to manufacturers’ protocols. The proteins were separated on 4–15% gradient SDS–polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Bellerica, MA). The following primary antibodies were used: anti-CK2α (Millipore), anti-Gli1 (Cell Signaling), and anti-GAPDH (Trevigen, Gaithersburg, MD). After being incubated with appropriate secondary antibodies, the antigen-antibody complexes were detected by using an ECL blotting analysis system (Amersham Pharmacia Biotech, Piscataway, NJ). Digital images were prepared using Adobe Photoshop CS3.
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