Whole cell protein extracts were obtained by lysing 2–5 × 106 cells with Laemmli sample buffer. Protein concentration was measured with the Micro BCATM Protein Assay Reagent kit (Thermo Scientific Pierce, Rockford, IL, USA). Protein extracts (25 μg) were subjected to reducing conditions before being subjected to electrophoresis on a polyacrylamide gel and then transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). One hour after blocking with 5% (w/v) non-fat milk in Tris-buffered saline with 0.1% Tween®-20, the membranes were incubated with the specific primary antibodies against BCL-2 (clone 124, #M0887, Dako, Denmark), BIM (clone C34C5, #2933, Cell Signaling, Danvers, MA, USA), cleaved caspase 3 (#9661, Cell Signaling), ERK2 (clone 1B3B9, #05-157, Millipore, Temecula, CA, USA), NOXA (clone 114C307, #ab13654, Abcam, Cambridge, UK), MCL-1 (clone S-19, #sc-819, Santa Cruz Biotechnology, Dallas, TX, USA), PHB1 (clone H-80, #sc-28259, Santa Cruz Biotechnology), PHB2 (anti-REA, #07-234, Millipore), PARP (#9542, Cell Signaling), PUMA (#4976, Cell Signaling), β-Actin (clone AC-15, #A5441, Sigma-Aldrich) and Tubulin (clone Ab-1, #CP06, Oncogene, Darmstadt, Germany). Antibody binding was detected using a secondary antibody conjugated to horseradish peroxidase and the enhanced chemiluminescence (ECL) detection system (GE Healthcare, Amersham Place, Buckinghamshire, UK).
β actin clone ac 15
Manufactured by Merck Group
Sourced in United States, United Kingdom, Japan
β-actin (clone AC-15) is a monoclonal antibody that specifically binds to the β-actin protein. β-actin is a highly conserved cytoskeletal protein found in all eukaryotic cells and is commonly used as a loading control in western blot analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (4 mentions)
Hybond, by Cytiva (4 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
Complete protease inhibitor cocktail, by Roche (3 mentions)
α tubulin clone dm1a, by Merck Group (3 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Enhanced chemiluminescence system, by GE Healthcare (2 mentions)
Hif 1α, by BD (2 mentions)
Precision plus protein dual color standards marker, by Thermo Fisher Scientific (2 mentions)
Phospho smad1 5 8, by Cell Signaling Technology (2 mentions)
Smad5 clone ep619y, by Abcam (2 mentions)
Clone 18 bmpr 2, by BD (2 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (2 mentions)
Phospho jnk, by Cell Signaling Technology (2 mentions)
Gapdh, by Merck Group (2 mentions)
Monoclonal mouse anti human desmin d33 antibody, by Agilent Technologies (2 mentions)
2 deoxy d 2 6 3h glucose, by Hartmann Analytic (2 mentions)
Vector vip hrp substrate kit, by Vector Laboratories (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
55 protocols using β actin clone ac 15
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Apoptosis Markers
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Proteins in whole-cell lysates were separated by SDS-PAGE and transferred to PVDF membranes that were probed using antibodies raised against: EBNA1 (AMo serum); EBNA2 (PE2); LMP1 (CS1-4); BHRF1 (5B11); β-actin (clone AC-15; Sigma); caspase-3, -7 and -9 (nos. 9662, 9492, 9502; all CST); DEDD2/FLAME3 (14574; Proteintech, Chicago, IL, USA); PUMA (D30C10; CST); BIM (no. 2819; CST); CFLAR (10394-1-AP; Proteintech); CASP8AP2 (PA5-19954; Life Technologies); BID (no. 2002; CST); BAD (no. 9292; CST); BCL-2 (C-2; Santa Cruz Biotechnology (SCBT, Dallas, TX, USA)); BCL-X (H-5; SCBT); MCL-1 (no. 4572; CST); cIAP1 (no. 4952; CST), Livin (D61D1; CST); Survivin (71G4B7; CST); XIAP (3B6; CST); NOXA (114C307; Abcam, Cambridge, UK); BAK (N-20; SCBT); and BAX (N-20; SCBT). Protein size analysis and densitometry were carried out using the Chemidoc MP system equipped with the ImageLab v.5.2 Software (Bio-Rad, Hercules, CA, USA). Size markers were SeeBlue Plus 2 (Life Technologies) and densitometry calculations were normalised to endogenous control proteins, calregulin or β-actin.
3
Erythroblast Protein Lysate Analysis
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Protein lysates from purified erythroblasts were prepared using cell lysis buffer with 1:500 protease inhibitor mixture (Sigma-Aldrich). Protein concentrations were determined using a Pierce bicinchoninic acid assay kit (Thermo Scientific, Rockford, IL, USA), and 10 μg of protein were resolved by 10% SDS-PAGE gel. After being transferred to polyvinylidene fluoride membranes (Whatman), proteins were detected using primary antibodies and HRP-conjugated secondary antibodies. The following antibodies for western blots were used: β-actin (clone AC-15, A3854, Sigma-Aldrich) and 14-3-3ζ (cat# sc-1019, Santa Cruz Biotechnology). Horseradish peroxidase-conjugated anti-β-actin antibody was used as a loading control. Western blots were developed using SuperSignal West Pico chemiluminescent reagent. Western blotting secondary antibodies, markers, and reagents were obtained from Thermo Scientific.
4
BMP9 Signaling Activation Assay
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Cells were stimulated in 0% FCS with recombinant BMP9 (R&D Systems) at the concentration indicated for 1 h after 1 h30 of serum deprivation. Cell extracts were lysed in 50 mmol/L Tris-HCl pH 7.4, 0.5 mol/L NaCl and a cocktail of protease inhibitors (Sigma) by sonication. 20 μg of proteins from cell lysates were separated on a SDS/PAGE, 4–20% (Bio-Rad) and analyzed by immunoblotting with anti-pSmad1/5/9 antibody (Cell Signaling #9511). The same membrane was reprobed with a monoclonal antibody against β-actin (clone AC-15; Sigma) to confirm equal protein loading.
5
Cell Lysis and Immunoblot Analysis
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Cell lysis, SDS‐PAGE and immunoblot analysis were performed as described previously.8 (link) Membranes were incubated with antibodies against the following proteins: BIG3 (diluted 1:250),6 (link) PHB2 (Abcam #ab71970, diluted 1:1000), β‐actin (clone AC‐15, Sigma‐Aldrich #A1978, diluted 1:5000), PARP (CST #9542, diluted 1:500), and apoptosis‐inducing factor (AIF) (clone E‐1, Santa Cruz Biotechnology #sc‐13116, diluted 1:200). After incubation with HRP‐conjugated secondary antibodies (anti‐mouse IgG‐HRP, diluted 1:5000; anti‐rat IgG‐HRP, diluted 1:5000; anti‐rabbit IgG‐HRP, diluted 1:1000 [Santa Cruz Biotechnology]) for 1 hour, the blots were developed with an enhanced chemiluminescence system (GE Healthcare) and scanned using an Image Reader LAS‐3000 Mini (Fujifilm).
6
Protein Expression Analysis by Immunoblotting
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Immunoblotting was performed essentially as described in a previous report [22 (link)]. We used the following antibodies as primary antibodies: p53 (clone DO-1, sc-126 HRP, Santa Cruz Biotechnology), p21 (clone EA10, Calbiochem), PUMA (Ab-1, Calbiochem), p53DINP1 (NB100-56627, Novus Biologicals), GAPDH (clone 6C5, GeneTex), β-Actin (clone AC-15, Sigma), caspase-3 (ab90437, abcam), or caspase-7 (clone 4G2, MBL, Japan).
7
Western Blot Analysis of Key Signaling Proteins
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Cells lysed in NP-40 buffer and equal amounts of protein were loaded in sodium dodecyl sulfate sample buffer onto a sodium dodecyl sulfate-polyacrylamide gel, blotted onto a polyvinylidene difluoride membrane, and analyzed for expression of either MMP-9 (1:500; R&D Systems AF911), p-AKT (1:1000; Cell Signaling 4060), pan AKT (1:1000; Cell Signaling 4691), GAPDH (1:7000; Cell Signaling 2118), INSR (1:500; Cell Signaling 74118), and Hif-1α (1:250; BD Biosciences Catalog number 610959). To detect the expression of β-Actin clone AC-15 (1:2000; Catalog Number A5441) from Sigma was used. Protein sizes were determined using the Precision Plus Protein Dual Color Standards marker (Catalog Number 161-0374) from Life Technologies (Carlsbad, CA, USA). The IGF-1R Ab (0.05 µg/µl final concentration) used was MK-0646 obtained was a kind gift from the PPTC59 . The recombinant human IGF1 and IGF2 were purchased from R&D biosystems (Cat. Number 291-G1 and 292-G2) (10 ng/ml final concentration). All blots were derived from the same experiment and were processed in parallel.
8
Immunoblotting of miRNA Targets and p27
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Cells were lysed in radioimmunoprecipitation (RIPA) buffer (50 mM Tris, pH 7.4, 1% TX-100, 0.2% Na deoxycholic acid, and 0.2% SDS, HALT complete tab [Roche]). Proteins were quantified using the Pierce BCA Protein Assay kit (ThermoFisher Scientific) and equal amounts of protein were run on 4–12% Bis-Tris gradient gels (Invitrogen) according to manufacturer’s instructions. Proteins were transferred by semi-dry electrophoresis (BioRad) onto Immobilon-P PVDF (EMD Millipore). Membranes were incubated overnight at 4°C with anti-p27 3688 (Cell Signaling Technologies) or HoxA7 ab70027 (Abcam), washed then incubated with anti-mouse or anti-rabbit HRP conjugated secondary antibodies (GE Healthcare). Membranes were developed on film with WesternSure Premium Chemiluminescent Substrate (LI-COR). The same Immunoblots were reprobed with β-actin clone AC-15 (Sigma) as loading control. Biotinylated miRNA target immunoblots and p27Kip1 immunoblots were repeated on lysates generated from at least two independent experiments each.
9
Western Blot Analysis of Signaling Pathways
10
Western Blot Analysis of Phospho-ERK
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Total proteins from T-ALL or MS5 cells were extracted with 1× lysis buffer, separated by 12% SDS–PAGE and transferred onto nitrocellulose membrane (GE Healthcare, Velizy-Villacoublay, France). Membranes were incubated with antibodies against phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (4376; Ozyme, Saint Quentin Yvelines, France), p44/42 MAPK (ERK1/2) (4695; Ozyme) and β-actin (clone AC-15; Sigma-Aldrich). Proteins were detected with the ECL PLUS chemiluminescent substrate (GE Healthcare).
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