20–30 µg protein were resolved on a 10% SDS-polyacrylamide gel, transferred onto a nitrocellulose membrane and probed with primary antibodies (anti-choline kinase alpha [Abcam; ab88053], anti-vinculin [Abcam; ab129002], anti-tubulin [Cell Signalling; 2128S], anti-E2F5 [Santa Cruz; sc-1083], anti-E2F1 [Cell Signalling; 3742], anti-α3+β1 [Abcam; ab217145], anti-HIF-1α [Protein Tech; 20960-1-AP], anti-HIF-2α [Bethyl Labs; A700–003]) at 4°C for 12 h. Membranes were incubated with anti-rabbit (1:10,000) horseradish peroxidase-linked secondary antibody (Cell Signalling) for 2 h at room temperature. Signal was developed using Amersham ECL Prime Western blotting Detection reagent (GE Healthcare, Chicago, IL, USA), and images taken using a G:BOX gel imaging system (Syngene, Cambridge, UK).
Ab129002
Manufactured by Cell Signaling Technology
Sourced in United States
Ab129002 is a primary antibody product offered by Cell Signaling Technology. It is a polyclonal antibody that targets a specific protein. The core function of this product is to detect and bind to the target protein in biological samples for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 710, by Zeiss (2 mentions)
Ab88053, by Abcam (1 mentions)
Anti e2f1, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase linked secondary antibody, by Cell Signaling Technology (1 mentions)
G box gel imaging system, by Syngene (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Phospho paxillin, by Cell Signaling Technology (1 mentions)
A21422, by Thermo Fisher Scientific (1 mentions)
A21429, by Thermo Fisher Scientific (1 mentions)
Phospho erm, by Cell Signaling Technology (1 mentions)
Phospho cofilin, by Cell Signaling Technology (1 mentions)
Vinculin, by Abcam (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Fv3000, by Olympus (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Ab37977, by Abcam (1 mentions)
Eif2α, by Cell Signaling Technology (1 mentions)
P p70s6k thr389, by Cell Signaling Technology (1 mentions)
Atf 4 d4b8, by Cell Signaling Technology (1 mentions)
5 protocols using ab129002
1
Western Blot Analysis of Protein Expression
2
Immunocytochemistry of Cell Cytoskeletal Proteins
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The U2OS cells, fibroblast BJ-hTERT cells or rat vascular SMCs were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 15 min and permeabilized with 0.1% Triton X-100 in PBS for 10 min. The fixed cells were incubated with a blocking solution (5% bovine serum albumin in PBS with 0.1% Tween20) at room temperature for 1 h. The cells were incubated with primary antibodies, vimentin (Santa Cruz Biotechnology, sc6260, 1:100), cytokeratin 14 (Abcam, ab7800, 1:250), vinculin (Abcam, ab129002, 1:500), phospho-Paxillin (Cell Signaling, 2541, 1:50), phospho-ERM (Cell Signaling, 3726, 1:200), or phospho-Cofilin (Cell Signaling, 3313, 1:100) in the blocking solution at 4°C overnight. Secondary antibodies (Thermo Fisher Scientific, A-21422, A-21429, 1:2000) were incubated at room temperature for 1 h in the blocking solution. Alexa Fluor488-phalloidin (Thermo Fisher Scientific, A12379, 1:1000) and Alexa Fluor555-β-tubulin (Abcam, ab206627, 1:1000) were also incubated at room temperature for an hour in the blocking solution. Images were captured using the confocal microscope FV3000 (Olympus, Tokyo) or LSM 710 (ZEISS). Captured images were analyzed by ImageJ (version 1.53d, National Institutes of Health (NIH), United States).
3
Quantifying PDK4 Protein Levels
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Whole-cell lysates were prepared in RIPA lysis buffer (Merck Millipore, Burlington, USA). Primary antibodies against PDK4 (human: 1:200; Abcam; ab38242; mice: 1:500; Novus Biologicals; NBP1-07047), vinculin (1:2000; Abcam; ab129002) and tubulin (Cell Signaling Technology; Denver; USA; 2148) were used. After development with an HRP-conjugated secondary antibody, semiquantitative analysis was performed applying ImageJ software (NIH). The quotient of areas under the curve (AUC) for PDK4 and housekeeping protein (AUC PDK4/AUC tubulin or vinculin) is representative of the amount of PDK4 protein present in the loaded sample. In humans, results from several western blots were pooled to the corresponding group (n = 8 patients in each group; lowest PDK4 transcripts versus highest PDK4 transcripts). In murine hepatocytes treated with increasing concentrations of fenofibrate, data from three independent experiments were pooled.
4
Immunoblotting analysis of U2OS cells
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For experiments where U2OS cells were exposed to compound for less than 24 h, 60,000 cells/well were seeded onto 12-well plates. For the experiments comparing cells growing in 10% FCS/starvation for all U2OS (30,000/60,000 cells/well), MCF7 and RPE (40,000/80,000 cells/well). RIPA buffer with protease and phosphatase inhibitors (Sigma, Germany) was used for preparing protein lysates from U2OS cells treated as indicated. Immunoblotting was performed following standard protocols with indicated antibodies: ATF4 (D4B8) (1:500 or 1:50, Cell Signaling Technology, 11815S), p-P70S6K (Thr389) (1:1,000, Cell Signaling Technology, 9205), 4E-BP1 (1:1,000, Cell Signaling Tech, 9452S), p-eIF2α (phospho S51) [E90] (1:1,000, Abcam, ab32157), eIF2α (1:500, Cell Signaling Technology, 9722), Vinculin (EPR8185) (1:2,000, Abcam, ab129002), PERK (D11A8) (1:500, Cell Signaling Technology, 5683), SPHK1(D1H1L) (1:500, Cell Signaling Technology, 12071S), SPHK2 (1:50, Abcam, ab37977), and GAPDH (1:100, Abcam, ab9485). Protein bands were visualized by chemiluminescence (either with ECL, Thermo Scientific, 34076, or Amersham ECL, GE Healthcare, RPN2235) and imaged on an Amersham Imager 600 (GE Healthcare, USA). In order to detect changes in SPHK2, and ATF4 simultaneously, samples were analyzed using JESS/WES immunoblot system (ProteinSimple, USA).
5
Immunoblotting of Mouse Tumor Samples
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Mouse tumors were isolated at necropsy and portions subjected to flash freezing in liquid N2 or fixed in formalin for paraffin-embedding. Frozen tumors were pulverized under liquid N2, sonicated in RIPA buffer and centrifuged to remove insoluble material. Supernatants were aliquoted and stored at -80 • C. Immunoblotting was performed as previously described [27] (link). Quantification of immunereactive bands on immunoblots was performed using ImageJ software (www.rsb.info.nih.gov/ij/). The area under curve of the specific signal was corrected for the AUC of actin. Immunoblot antibodies: anti ErbB2 (abcam #ab2428-1); Actin (Sigma #A-2066); vinculin (abcam #ab129002); IKK␣ (Cell Signaling #2682).
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