Analysis of protein levels was performed using western blotting. Cell lysates from different treatment groups were prepared as described previously [14 (link), 15 (link)]. Proteins (25–40 μg protein) were electrophoresed on premade 10% Trisglycine gels (Invitrogen) and then transferred onto nitrocellulose membranes. After blocking in freshly prepared PBS containing 3% non-fat dry milk at room temperature for 30 min, the membranes were incubated with antibodies against DNMT1, DNMT3a and DNMT3b, CDKN1A, CDC2, pCDC2, and GADPH at 4 °C overnight followed by an anti-rabbit peroxidase-conjugated secondary antibody at 1:1000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA). Protein bands were visualized on X-ray film using an enhanced chemiluminescence system (Amersham Life Science, Piscataway, NJ). Equal protein loading was verified using anti-b actin antibody. Experiments were repeated three times, and thus three western blots were run in each experiment, and representative blot is shown in each case.
Anti rabbit peroxidase conjugated secondary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The Anti-rabbit peroxidase-conjugated secondary antibody is a laboratory reagent designed for use in various immunoassay techniques. It is a polyclonal antibody that specifically targets rabbit primary antibodies and is conjugated to the enzyme peroxidase. This secondary antibody is used to detect and visualize the presence of rabbit primary antibodies in samples.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti actin, by Merck Group (2 mentions)
Horseradish peroxidase conjugated anti mouse secondary antibody, by Merck Group (2 mentions)
Tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence system, by Cytiva (1 mentions)
Diaminobenzidine colorimetric reagent solution, by Agilent Technologies (1 mentions)
Phospho acc ser79, by Cell Signaling Technology (1 mentions)
Phospho ampk thr172, by Cell Signaling Technology (1 mentions)
Image scop software, by Media Cybernetics (1 mentions)
Rabbit anti nav1, by Alomone (1 mentions)
Antimouse horseradish peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Anti nav1, by Alomone (1 mentions)
Cell extraction buffer, by Thermo Fisher Scientific (1 mentions)
Anti stat3 ab68153, by Abcam (1 mentions)
Anti β actin sab5500001, by Merck Group (1 mentions)
Hepg2, by Genechem (1 mentions)
Molecular weight protein standards, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Antibody against gapdh, by Santa Cruz Biotechnology (1 mentions)
Ecl western blot system, by GE Healthcare (1 mentions)
11 protocols using anti rabbit peroxidase conjugated secondary antibody
1
Western Blot Analysis of Protein Levels
2
Immunohistochemical Analysis of Xenograft Tumors
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The xenograft tumor slides were incubated with the following primary antibodies: phospho-AMPK (Thr172) (1:100), phospho-ACC (Ser79) (1:100) (Cell Signaling Technology, USA). Anti-rabbit peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) and diaminobenzidine colorimetric reagent solution purchased from Dako (Carpinteria, CA) was used. The staining process were according to standard methods. Assessment of the staining was performed using the Image-scop software (Media Cybernetics, Inc.) according to the staining intensities and the percentage of positively stained cells, as described [53 (link)].
3
Western Blot Analysis of CBS and Nav1.7
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Expressions of CBS and Nav1.7 in T13-L2 DRGs from PMS and CON rats were determined using Western Blot analysis, as previously described in detail.27 (link) The primary antibodies including rabbit anti-CBS (1:1000, Abnova, USA), rabbit anti-Nav1.7 (1:1000, Alomone, USA), or mouse anti-β-actin (1:1000, Sigma, USA) and secondary antibodies including antirabbit peroxidase-conjugated secondary antibody (1:2000; Santa Cruz Biotechnology, CA) and antimouse horseradish peroxidase-conjugated secondary antibody (1:4000, Sigma, USA) were used to probe the target proteins.
4
Quantifying NaV1.7 and NaV1.8 in Diabetic Rats
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Expressions of NaV1.7 and NaV1.8 in colonic DRGs (T13-L2 DRGs) from diabetic and CON rats were determined using western blotting analysis, as previously described in detail.15 The primary antibodies used to probe the target proteins included rabbit anti-NaV1.7 or anti-NaV1.8 (1:200, Alomone Labs, Jerusalem, Israel), rabbit anti-GAPDH (1:1000, Biotechnology Co., CHN), and mouse anti-actin (1:1000; Chemicon, Temecula, CA, USA). The secondary antibodies included anti-rabbit peroxidase-conjugated secondary antibody (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-mouse horseradish peroxidase-conjugated secondary antibody (1:4000; Chemicon).
5
Western Blot Analysis of P2X2 and P2X3 in Diabetic Rats
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Expressions of P2X2 and P2X3 in T13-L2 DRGs from diabetic and CON rats were determined using western blotting analysis, as previously described in details19 (link), 35 (link). The primary antibodies including rabbit anti-P2XRs (1:1000, Abcam, USA), rabbit anti-GAPDH (1:1000, Biotechnology Co., CHN), mouse anti-actin (1:1000; Chemicon, Temecula, CA) and the secondary antibodies including anti-rabbit peroxidase-conjugated secondary antibody (1:2000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse horseradish peroxidase-conjugated secondary antibody (1:4000; Chemicon) were used to probe the target proteins.
6
Gefitinib and GSK126 Modulate Signaling Pathways
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MGC803 cells were treated with Gefitinib and/or GSK126 for 48 hours. Total protein was extracted using Cell Extraction Buffer (Biosource, Camarillo, CA, USA) supplemented with protease and phosphatase inhibitors. Western blotting was routinely conducted.14 (link) In brief, protein lysates were separated by SDS-PAGE and transferred to nitrocellulose membrane, followed by incubation with the rabbit-derived primary antibodies against phosphorylated protein kinase B (Akt), phosphorylated mammalian target of rapamycin (mTOR), phosphorylated S6, phosphorylated Unc-51-like autophagy activating kinase (ULK), and microtubule-associated protein 1A/1B-light chain 3 (LC3) (1:1,000, all from Cell Signaling Technology, Danvers, MA, USA). After being stripped, the membranes were reprobed with antibodies against total Akt, mTOR, S6, ULK, and GAPDH (1:1,000, all from Cell Signaling Technology). The peroxidase-conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used for the development of signals, which were visualized with ECL reagent (Pierce, Rockford, IL, USA). GAPDH was used as the internal standard of total target proteins, and phosphorylated proteins were normalized to their total proteins, respectively. Signals from triplicates (n=3) were quantified by image software ImageJ (NIH, Bethesda, MD, USA) and the intensity of the control was set to 1.
7
Hepatocellular Carcinoma Cell Lines
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Materials and cell lines. HepG2 and SMCC-7721 cell lines, which originate from primary hepatoblasts (19) and mature hepatocytes, respectively, were used in the present study. The human HB cell line HepG2 and the HCC cell line SMCC-7721 were obtained from GeneChem Co. Ltd. (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS), in an atmosphere of 5% CO 2 at 37˚C. Polyclonal anti-σ1R (AP2747A; Abgent, San Diego, CA, USA), anti-NF-κB (8242S; Cell Signaling Technology, Inc., Danvers, MA, USA), anti-STAT-3 (Ab68153; Abcam, Cambridge, UK), and anti-β-actin (SAB5500001; Sigma-Aldrich ® Co. LLC, St. Louis, MO, USA) antibodies were used for immunohistochemistry or western blotting. The peroxidase-conjugated anti-rabbit secondary antibody was purchased from Santa Cruz Biotechnology, Inc. (sc-2004; Santa Cruz, CA, USA).
8
Western Blot for Alkaline Phosphatase Quantification
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After treatments, cells were washed and lysed as previously described [34 (link)]. Twenty micrograms of total protein extract were separated by 12% SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (GE Healthcare Milano, Italy) in a cooling system at 100 V for 1 h. Membranes were saturated for 1 h at room temperature with 0.1% Tween-20 (Sigma-Aldrich), 5% dry milk (Bio-Rad Laboratories, Hercules, CA) in PBS. Membranes were then incubated with an antibody against human tissue non-specific alkaline phosphatase (Abcam, Cambridge, UK) diluted 1:10,000, overnight at 4°C, washed several times and incubated with peroxidase-conjugated secondary anti-rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1:10,000, for 1 h at room temperature. Specific bands were then detected by ECL Western blot system (GE Healthcare). Antibody against GAPDH (Santa Cruz Biotechnology) were used to perform a normalizing control. Densitometry of bands was performed with ImageJ software [http://rsbweb.nih.gov/ij/download.html ]. Molecular sizes were evaluated referring to protein molecular weight standards (Bio-Rad Laboratories). Each treatment and analysis was performed at least in triplicate separate experiments.
9
Detecting GPR68 Expression in Cell Lines
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Human embryonic kidney 293 cells (HEK-293; DMSZ, Braunschweig, Germany), either mock-transfected or transfected with human GPR68, or BON-1 cells (DMSZ, Braunschweig, Germany) endogenously expressing GPR68 were seeded onto poly-L-lysine-coated 60-mm dishes and grown to 80% confluency. Cells were lysed in detergent buffer (20 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10% glycerol, 0.1% SDS, 0.2 mM phenylmethylsulfonylfluoride, 10 mg/mL leupeptin, 1 mg/mL pepstatin A, 1 mg/mL aprotinin, and 10 mg/mL bacitracin). GPR68 was enriched using wheat germ lectin agarose beads. Samples were subjected to 7.5% SDS-polyacrylamide gel electrophoresis and immunoblotted onto polyvinylidene fluoride (PVDF) membranes. Blots were incubated with the rabbit monoclonal anti-GPR68 antibody 16H23L16 at a dilution of 1:1000 followed by 1:5000-diluted peroxidase-conjugated secondary anti-rabbit antibody (Santa Cruz Biotechnology, Dallas, TX, USA) and enhanced chemiluminescence detection (Amersham, Braunschweig, Germany).
When indicated, expression of endogenous GPR68 in BON-1 cells was silenced using chemically synthesised double-stranded GPR68 siRNA duplexes (Santa Cruz Biotechnology, Dallas, TX, USA) according to the manufacturer’s instructions. As negative control, scrambled siRNA was used (Santa Cruz Biotechnology, Dallas, TX, USA).
When indicated, expression of endogenous GPR68 in BON-1 cells was silenced using chemically synthesised double-stranded GPR68 siRNA duplexes (Santa Cruz Biotechnology, Dallas, TX, USA) according to the manufacturer’s instructions. As negative control, scrambled siRNA was used (Santa Cruz Biotechnology, Dallas, TX, USA).
10
VPAC1 and VPAC2 Receptor Expression Analysis
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Stably transfected HEK 293 cells were seeded onto poly-l -lysine-coated 60 mm dishes and grown to 80% confluence. The cells as well as freshly dissected tissues from WT (C57BL6) and VPAC2-deficient mice, including small and large intestine, gastric mucosa, brain, pancreas, spleen and testis, were lysed in detergent buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 10 mM disodium pyrophosphate, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS) in the presence of protease and phosphatase inhibitors Complete Mini and PhosSTOP (Roche Diagnostics). The receptors were enriched using wheat germ lectin agarose beads as described previously (29, 30) (link). Proteins were eluted from beads using SDS sample buffer for 20 min at 45 °C. The samples were then subjected to 7.5% SDS–polyacrylamide gels, and blotted onto PVDF membranes. The membranes were incubated with the rabbit monoclonal anti-human VPAC1 antibody (SP234; dilution 1:500) or the rabbit monoclonal anti-human VPAC2 antibody (SP235; dilution 1:500), followed by incubation with peroxidase-conjugated secondary anti-rabbit antibody (Santa Cruz Biotechnology; dilution 1:5000) and ECL detection (Amersham). For adsorption controls, antibodies were preincubated with 10 μg/ml of their cognate peptides for 2 h at room temperature.
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