Cells were lysed in AM1 lysis buffer (Active Motif, Carlsbad, CA) and protein concentrations were measured with the BCA protein assay kit (Thermo Fisher Scientific Inc., Carlsbad, CA). Total protein (50 μg) was resolved by 125 g/L SDS-PAGE and transferred onto PVDF membranes. After being blocked in TBST (20 mmol/L Tris, 137 mmol/L NaCl, 1 g/L Tween20, pH 7.6) with 50 ml/L skim milk for 2 h at room temperature, membranes were incubated with CD44, CD133 (Miltenyi Biotech, San Diego, CA), Oct4 (Cell Signaling Technology, Danvers, MA), Nanog (Santa Cruz Biotechnology, Santa Cruz, CA), β-catenin (Cell Signaling Technology), and SOX2 (Cell Signaling Technology), and β-actin primary antibodies (diluted 1:500; Santa Cruz Biotechnology) for 2 h. Membranes were then washed three times with TBST solution, followed by incubation for 1 h with HRP-linked secondary antibodies (1:1000; Santa Cruz Biotechnology) at room temperature. Finally, membranes were visualized using the DAB reagent (Dako Corporation, Carpinteria, CA).
β actin primary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
The β-actin primary antibody is a tool used in research laboratories to detect and measure the presence of the β-actin protein. β-actin is a widely expressed cytoskeletal protein that plays a fundamental role in cellular structure and function. This antibody can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and immunocytochemistry, to study the expression and localization of β-actin in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (5 mentions)
Bca experiment, by Beyotime (4 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (3 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (3 mentions)
Antibody against anp32e, by Abcam (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
α mem, by Thermo Fisher Scientific (2 mentions)
Luciferase assay kit, by Promega (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Psv β galactosidase vector, by Promega (2 mentions)
Cleaved caspase 9, by Santa Cruz Biotechnology (2 mentions)
Second antibody, by Jackson ImmunoResearch (2 mentions)
Cleaved caspase 3, by Santa Cruz Biotechnology (2 mentions)
Dab reagent, by Agilent Technologies (1 mentions)
Hrp linked secondary antibody, by Santa Cruz Biotechnology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Nanog, by Santa Cruz Biotechnology (1 mentions)
19 protocols using β actin primary antibody
1
Western Blot Analysis of Stem Cell Markers
2
Immunomodulatory Effects of Calcineurin Inhibition
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RPMI-1640, fetal bovine serum (FBS), glutamine, penicillin, streptomycin, and 4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid were purchased from Gibco (Grand Island, NY, USA). Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China). FK506, MFN2, and β-actin primary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Methyl-thiazolyl-tetrazolium (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits for IL-2, IL-4 and interferon (IFN)-γ were obtained from Biosource (Worcester, MA, USA). Fluo-3/AM and pluronic F-127 were obtained from Molecular Probes (Eugene, OR, USA). TRIzol reagent was obtained from Invitrogen (Carlsbad, CA, USA). Total RNA isolation and reverse transcription systems were purchased from Promega (Madison, WI, USA). The Biomol Green Calcineurin Assay kit was purchased from Biomol (Plymouth Meeting, PA, USA). Nuclear extract and TransAM NFAT kits were obtained from Active Motif (Carlsbad, CA, USA). Nondenaturing lysis buffer and protease inhibitor cocktail were purchased from Applygen Technologies Inc., (Beijing, China). An Amersham enhanced chemiluminescence (ECL) Advance Western Blotting Detection kit was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden).
3
Resistin-Dependent Signaling Pathway Regulation
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The recombinant human resistin was purchased from R&D Systems (Minneapolis, MN, USA). We purchased p85, Akt, and β-actin primary antibodies (Santa Cruz Biotechnology, CA, USA), as well as rabbit polyclonal antibodies specific for p-p85 and p-Akt (Cell Signaling Technology, Danvers, MA, USA). The miR-16-5p mimic, miRNA control, Lipofectamine 2000, and Trizol were purchased from Life Technologies (Carlsbad, CA, USA). Dharmacon Research (Lafayette, CO, USA) supplied ON-TARGETplus siRNAs. Gibco-BRL life technologies (Grand Island, NY, USA) supplied fetal bovine serum (FBS), DMEM, α-MEM, and all other cell culture reagents. Promega (Madison, WI, USA) supplied the pSV-β-galactosidase vector and luciferase assay kits. All other chemicals or inhibitors were purchased from Sigma-Aldrich (St. Louis, MO, USA).
4
Signaling Pathways Modulation by CCN6
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We purchased p85α, Akt1, p-IKKα/β (Ser180/Ser181), p-IκBα, p65 (Ser536), IKKα/β, IκBα, p65, CCN6, and β-actin primary antibodies (Santa Cruz Biotechnology, CA, USA), and rabbit polyclonal antibodies specific for p-p85 (Y458), p-Akt (S473), p-mTOR (Ser2448), and mTOR (Cell Signaling Technology, Danvers, MA, USA). Recombinant human CCN6 was purchased from PerpoTech (Rocky Hill, NJ, USA) and CCN6 short hairpin (sh)RNA expression plasmids from RNAiCore (Taipei, Taiwan). The D-Luciferin potassium salt was purchased from Gold Biotechnology (St. Louis, MO, USA). Lipofectamine 2000 and TRIzol were purchased from Life Technologies (Carlsbad, CA, USA). Dharmacon Research (Lafayette, CO, USA) supplied ON-TARGETplus siRNAs. Gibco-BRL life technologies (Grand Island, NY, USA) supplied DMEM, α-MEM, fetal bovine serum (FBS), and all other cell culture reagents. The Matrigel was purchased from BD (Biosciences, Bedford, MA, USA) and Promega (Madison, WI,) supplied the pSV-β-Galactosidase Vector and luciferase assay kits. All other USA chemicals or inhibitors that we used were supplied by Sigma-Aldrich (St. Louis, MO, USA).
5
Western Blot Analysis of Cellular Signaling Pathways
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Tissues were lysed for 30 min prior to centrifugation at 1,204 × g and 4°C for 5 min. The protein concentration in supernatant was determined using the bicinchoninic acid method using a BCA kit (Biotek China, Beijing, China). Total protein (10 µg per lane) was subjected to 10% SDS-PAGE at a constant 80 V for 30 min followed by 120 V until the markers (Takara Biotechnology Co., Ltd.) reached the edge of the gel. The samples were electrotransferred onto a polyvinylidene difluoride membrane (Takara Biotechnology Co., Ltd.) at constant 80 V at 4°C for 2 h. The membrane was blocked with skimmed milk (1%) for 1 h at room temperature. Mouse anti-human RAF, AKT, ERK, IκB-α, p105/P50, RAS, P-AKT, FAK, MEK, p65 (1:1,000 dilution) and β-actin primary antibodies (1:5,000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) were added, followed by incubation with shaking overnight at 4°C. After rinsing with PBS containing Tween-20 (3×10 min), the rabbit anti-mouse monoclonal secondary antibody (1:3,000; Santa Cruz Biotechnology, Dallas, TX, USA) labeled with horseradish peroxidase was added prior to incubation at room temperature for 2 h, followed by rinsing with PBS containing Tween 20 (3×10 min). The immunoreactive bands were visualized by enhanced chemiluminescence (ECL enhanced chemiluminescence kit; Takara Biotechnology Co., Ltd.).
6
Cellular Stress Response Mechanisms
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WA was purchased from Alexis Biochemicals, USA. TritonX-100, FBS, DMEM, trypsin-EDTA solution, amphotericin B, penicillin and streptomycin were obtained from Hi-Media, Mumbai, India. DMSO, Wortmannin, Trypan blue, TMRE, Bradford reagent, Luminol, Cycloheximide, Z-vad-fmk, H2DCFDA, NAC, P-formaldehyde, PI and HRP-conjugated anti-GAPDH antibody were from SIGMA-Aldrich, USA. Ascorbic acid was purchased from Merck, (Darmstadt, Germany). Cell lysis buffer was purchased from BD Pharmingen, USA. Annexin V-FITC apoptosis detection kit was from Cayman Chemicals, USA. Developer, fixer and X-ray films were from KODAK, Japan. Agar100 resin was obtained from Agar Scientific Co., UK. Trizol was purchased from Invitrogen (CA, USA). All reagents for RT-PCR were from Fermentas (Burlington, Canada). Other chemicals were of analytical grade and purchased locally. β-actin primary antibody and HRP conjugated anti-mouse, anti-rabbit secondary antibodies were obtained from Santacruz, USA. AIP1/Alix and PCNA antibodies were obtained from BioLegend, USA, GRP-78, CHOP antibodies from Cell-Signaling, USA.
7
Protein Expression Analysis via Western Blot
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Western blotting was carried out by transfering whole cell protein from 10% SDS‐PAGE onto the PVDF membranes (Millipore) and incubating the membranes with the primary and secondary antibodies. Primary antibodies against PRKD3, p‐ERK1/2 (Thr202/Tyr204), ERK1/2, p‐c‐MYC (Ser62) and c‐MYC were purchased from Cell Signalling Technology. β‐actin primary antibody, Anti‐rabbit and antimouse secondary antibody were purchased from Santa Cruz Biotechnology.
8
Kidney Protein Immunoblotting Protocol
9
Analysis of Cellular Responses to Volasertib and miR-34a
10
Western Blot Analysis of CHI3L1 Protein
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Cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) with 2 mM phenylmethanesulfonyl fluoride (PMSF, Solarbio, Beijing, China) on ice. The proteins of the cell lysates were separated on a 10% SDS-polyacrylamide gel (SDS-PAGE) and then electrotransferred to a nitrocellulose membrane (GE Healthcare, Waukesha, WI, USA) in transferring buffer (25 mM Tris-HCl, 190 mM glycine, 20% methanol). After blocking with 5% non-fat milk in TBST buffer (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 8.0) for 90 min at room temperature, the membrane was incubated with a primary antibody in TBST buffer containing 1% non-fat milk overnight at 4°C. Subsequently, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (BioRad, Hercules, CA, USA) for 2 h at room temperature. The membrane was developed using a Western Lightning Plus-ECL kit (Perkin Elmer, Waltham, MA, USA). The CHI3L1 primary antibody was purchased from Abcam (Cambridge, MA, USA). The β-actin primary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
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