Collected cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with phenylmethanesulfonyl fluoride (PMSF, 1mM), both from Cell Signaling Technology (Danvers, MA, USA), and protease and phosphatase inhibitor cocktails (Roche Diagnostics, Mannheim, Germany). SDS-PAGE and Western blotting were performed according to the previously described protocol [37 (link)]. Staining of membranes with anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to assess equal sample loading. Antibodies against cyclin E1 (Clone: HE12, Cat. #: sc-247) and p-Rb [Ser 807/811] (sc-16670-R) were purchased from Santa Cruz Biotechnology. Antibody against cyclin B1 (ab2949) was purchased from Abcam (Cambridge, UK), cyclin D1 (92G2, 2978), c-Myc (D84C12, 5605), PARP (9542), Src (36D10, 2109), p-Src [Tyr 416] (D49G4, 6943), STAT3 (79D7, 4904), and p-STAT3 [Tyr 705] (D3A7, 9145) were purchased from Cell Signaling Technology.
Ab2949
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab2949 is a product offered by Abcam, a leading provider of life science research tools and reagents. It is a laboratory equipment item, but a detailed description without extrapolation or interpretation is not available.
Automatically generated - may contain errors
Lab products found in correlation
Bca assay, by Thermo Fisher Scientific (2 mentions)
Sds page, by Bio-Rad (2 mentions)
Blocking one, by Nacalai Tesque (2 mentions)
Ab9485, by Abcam (2 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
Sc 47778 hrp, by Santa Cruz Biotechnology (1 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
Sc 596, by Santa Cruz Biotechnology (1 mentions)
Sds page gel, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Ab32072, by Abcam (1 mentions)
Enhanced chemi luminescence (ecl), by Bio-Rad (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
A21422, by Thermo Fisher Scientific (1 mentions)
Fluorchem 8900 imager, by Bio-Techne (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Ab25901, by Abcam (1 mentions)
9 protocols using ab2949
1
Immunoblotting analysis of cell signaling
2
Western Blot Analysis of Cell Signaling
3
Fixation and Fluorescent Labeling of Oocytes
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Oocytes were fixed for 30–60 min at 37 °C in 100 mM HEPES (pH7; titrated with KOH), 50 mM EGTA (pH7; titrated with KOH), 10 mM MgSO4, 2% formaldehyde (methanol free) and 0.2% Triton X-100. Oocytes were left in PBS (phosphate-buffered saline) with 0.1% Triton X-100 overnight at 4 °C. F-actin staining was carried out in PBS with 0.1% Triton X-100 and 3% bovine serum albumin with Alexa-Fluor-488–Phalloidin (Molecular Probes, A12379; 1:100). DNA was stained with 5 mg ml−1 Hoechst 33342 or DAPI (Molecular Probes). As primary antibodies, we used mouse Cyclin B (Abcam, ab2949; 1:100). As secondary antibodies, we used Alexa-555-labelled goat anti-mouse (Life technologies, A21422; 1:500).
4
Western Blot Quantification of Cyclin B1
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To detect the cyclin B1 protein, cells were lysed in a lysis buffer. The total protein concentrations of the lysates were measured using bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL, USA). Cell lysates containing equal amounts of protein were separated by SDS-PAGE (Bio-Rad, Hercules, CA, USA), and protein bands were transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with Blocking One (Nacalai Tesque, Inc., Kyoto, Japan) overnight at 4 °C and then incubated for 1 h at 22–25 °C with anti-cyclin B1 antibody (ab2949; Abcam, Cambridge, UK) or an anti-glyceraldehyde 3-phosphate dehydrogenase antibody (ACR001PT; Acris Antibodies, Inc., San Diego, CA, USA) at a 1:1000 and 1:10,000 dilution, respectively, in a blocking solution. After washing three times, the membranes were incubated for 1 h at 22–25 °C with a secondary antibody (horseradish peroxidase-conjugated species-specific antibody). Immunoreactive bands were visualized with ImmunoStar LD (Wako Pure Chemical Industries). Western blotting chemiluminescence was quantified using an Alpha Innotech Fluorchem 8900 Imager and AlphaEase Software version 3.2.3.
5
Evaluation of Nitro-Substituted Hydroxynaphthanilides
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The tested nitro-substituted hydroxynaphthanilides 1 −6 were prepared and supplied by the Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic. The synthesis and structural characterization of these compounds have been described previously [3 (link),5 (link)]. Due to poor solubility in water, the compounds were dissolved in dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA), while the stock solutions were prepared freshly before each experiment. The final concentration of DMSO in the assays never exceeded 0.1% (v/v). Cisplatin and camptothecin were purchased from Sigma-Aldrich. RPMI 1640 and DMEM culture media, phosphate-buffered saline (PBS), foetal bovine serum (FBS) and antibiotics (penicillin and streptomycin) were obtained from HyClone Laboratories, Inc. (GE Healthcare, Logan, UT, USA). Mouse monoclonal antibodies against cyclin E1 (sc-247), caspase 3 (sc-7272) and caspase 9 (sc-17784) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibodies against cyclin B1 (ab2949) and caspase 8 (ab-25901) were purchased from Abcam (Cambridge, UK). All other reagents, unless specified elsewhere, were purchased from Sigma-Aldrich.
6
Western Blot Analysis of Cell Cycle Regulators
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Total protein was extracted using Pierce lysis buffer (Pierce, Rockford, IL). Protein quantification was performed using the Bradford method (Bio-Rad Co., USA). Proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in Tris buffered saline tween (TBST) with low-fat milk and then incubated overnight with primary antibodies against PLK1 (1:1000, ab109777, Abcam, USA), BUB1B (1:1000, ab70544, Abcam, CA, USA), CCNB1 (1:1000, ab2949, Abcam, CA, USA), CDC25A (1:1000, ab989, Abcam, CA, USA), FBXO5 (1:1000, ab129905, Abcam, CA, USA), NDC50 (1:3000, SAB1410085, Sigma, CA, USA) and GAPDH (1:2000, ab9485, Abcam, CA, USA) at 4 °C. The membranes were then washed with TBST and incubated with the horseradish peroxidase-conjugated secondary antibody goat anti-rabbit IgG (1:5000, Sigma, CA, USA). The blots were developed with ECL solution (Pierce, Rockford, IL, USA) and detected using a chemiluminescence system (Bio-Rad, CA, USA). Image Lab software was employed to analyze the intensities of the band signals obtained.
7
Immunohistochemical Analysis of Lung Tissue
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Left lung samples (4 µm) were washed in water for 15 min following dewaxing (xylene for 5 min; xylene and ethanol (1:5) for 5 min; xylene and ethanol (1:1) for 5 min) and incubated with 3% H2O2 for 10 min all at room temperature. Following the antigen was repaired at 95°C for 20 min and the slices were blocked by 0.1% goat serum (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at room temperature for 15 min. Sections were subsequently incubated with primary antibodies against Ki67 (ab15580, 1:100), p27Kip1 (ab7961, 1:100) and cyclin B1 (ab2949, 1:100; all provided by Abcam, Cambridge, MA, USA) at 4°C overnight, followed by incubation with second antibody (1% biotin-labeled goat anti-mouse IgG; ab47844; Abcam) at 37°C for 15 min. This was followed by incubation with horseradish peroxidase-labeled streptomyces ovalbumin at 37°C for 15 min and staining with diaminobenzidine (DAB) for 10–30 min at room temperature. The immunohistochemical results were analyzed using Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA) and semi-quantified to obtain integrated optical density (IOD) values. For each animal, 20 random visual fields of view were observed using an optical microscope (magnification, ×400) and an average IOD value calculated.
8
Western Blot Detection of Cyclin B1
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To detect the cyclin B1 protein, cells were lysed in a lysis buffer. The total protein concentrations of the lysates were measured using bicinchoninic acid (BCA) assay (Pierce Biotechnology, Rockford, IL, USA). Cell lysates containing equal amounts of protein were separated by SDS-PAGE (Bio-Rad, Hercules, CA, USA), and protein bands were transferred to polyvinylidene di uoride membranes (Bio-Rad). The membranes were blocked with Blocking One (Nacalai Tesque, Inc., Kyoto, Japan) overnight at 4°C and then incubated for 1 h at 22°-25°C with anti-cyclin B1 antibody (ab2949; Abcam, Cambridge, UK) or an anti-glyceraldehyde 3-phosphate dehydrogenase antibody (ACR001PT; Acris Antibodies, Inc., San Diego, CA, USA) at a 1:1000 and 1:10,000 dilution, respectively, in a blocking solution. After washing three times, the membranes were incubated for 1 h at 22°C-25°C with a secondary antibody (horseradish peroxidaseconjugated species-speci c antibody). Immunoreactive bands were visualized with ImmunoStar LD (Wako Pure Chemical Industries).
9
Western Blot Analysis of Protein Expression
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Radio-Immunoprecipitation Assay lysis buffer (Thermo Scientific) was used to extract total protein, which was obtained from tissues or cultured cells. The concentrations of proteins were determined using BCA protein kit (Beyotime, Shanghai, China). Next, proteins were subjected to 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore). Five percent of milk PBS with 0.1% Triton X-100 was used to block the membranes, which were then incubated with anti-CCNB1 antibody (ab2949, 1:1000, Abcam), antiactive-caspase-3 antibody (ab2302, 1:1000, Abcam), anti-CyclinD1 antibody (ab134175, 1:10000, Abcam), anti-TIMP-2 antibody (ab180630, 1:500, Abcam), anti-N-cadherin antibody (ab18203, 1:1000, Abcam) and anti-glyceraldehyde-3-phosphate dehydrogenase antibody (ab9485, 1:2500, Abcam) overnight at 4°C. After having been washed by Tween-20 for three times, the membranes were then incubated with appropriate horseradish peroxidase-conjugated secondary antibody (Protein tech). Protein bands were detected with enhanced chemiluminescence (Thermo Fisher Scientific) and visualized using Quantity one (Bio-Rad).
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