Western blot analysis was performed as described previously [6 (link)]. In brief, proteins from the CAMs or dissected coronary arteries were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins of these samples were then electrophoretically transferred onto a PVDF membrane (Millipore, USA). The membrane was blocked with 5% nonfat milk and then probed with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-labeled IgG (1:5000). Primary antibodies used were mouse anti-vimentin (1:1000, Abcam), rabbit anti-α-smooth muscle actin (SMA, 1:1000, Abcam), rabbit anti-calponin (1:1000, Abcam), rabbit anti-p62 (1:200, Abcam), mouse anti-ubiquitin (1:1000, Cell Signaling), rabbit anti-CDK1 (1:1000, Cell Signaling) or goat anti-β-actin (1:2000, Santa Cruz). The immuno-reactive bands were detected by chemiluminescence methods and visualized on Kodak Omat X-ray films. Densitometric analysis of the images obtained from X-ray films was performed using the Image J software (NIH).
Rabbit anti cdk1
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-CDK1 is a primary antibody that detects Cyclin-Dependent Kinase 1 (CDK1), also known as Cell Division Control Protein 1 (CDC2). CDK1 is a serine/threonine-protein kinase that plays a crucial role in the regulation of the cell cycle.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti calponin, by Abcam (4 mentions)
Mouse anti ubiquitin, by Cell Signaling Technology (4 mentions)
Rabbit anti p62, by Abcam (4 mentions)
Mouse anti vimentin, by Abcam (4 mentions)
Omat x ray film, by Kodak (2 mentions)
Goat anti β actin, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti α smooth muscle actin sma, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Rabbit anti α sma, by Abcam (2 mentions)
Fluoview fv1000, by Olympus (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Mouse anti pp2ac, by Santa Cruz Biotechnology (1 mentions)
Mouse anti b2m, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti p21, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemi luminescence (ecl), by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
5 protocols using rabbit anti cdk1
1
Western Blot Analysis of Vascular Markers
2
Immunocytochemistry Analysis of Cultured CAMs
3
Western Blot Analysis of CDK1, p21, and PP2Ac
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Rabbit anti-CDK1 was purchased from Cell Signaling Technology (MA, USA). Rabbit anti-p21, mouse anti-PP2Ac, and mouse anti-B2M antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Total protein was extracted using a lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, supplemented with protease inhibitors (10 mg/mL leupeptin, 10 mg/mL aprotinin, 10 mg/mL pepstatin A, and 1 mM 4-[2-aminoethyl] benzenesulfonyl fluoride). The protein extract was loaded onto an SDS-polyacrylamide gel, size-fractionated by electrophoresis, and then transferred to polyvinylidene fluoride (PVDF) membranes (Bio-Rad Laboratories). After blocking in 5% non-fat milk for 1 h, the membranes were incubated overnight with primary antibodies at 4°C. The protein expression was determined using horseradish peroxidase-conjugated antibodies followed by enhanced chemiluminescence (ECL, Millipore, St Charles, MO, USA) detection. The intensity of the bands was captured by JS-1035 image analysis scanning system (Peiqing Science & Technology, Shanghai, China). B2M was used as the internal control.
4
Immunocytochemistry Analysis of Cultured CAMs
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Cultured CAMs were grown on glass coverslips, stimulated or left unstimulated and fixed in 4% paraformaldehyde in phosphate-buffer saline (PFA/PBS) for 15 min. After permeabilization with 0.1% Triton X-100/PBS, the cells were rinsed with PBS and incubated overnight at 4°C with indicated primary antibodies: mouse anti-vimentin (1:200, Abcam), rabbit anti-α-SMA (1:200, Abcam), rabbit anti-calponin (1:200, Abcam), rabbit anti-p62 (1:200, Abcam), mouse anti-ubiquitin (1:200, Cell Signaling) and rabbit anti-CDK1 (1:200, Cell Signaling). The slides were extensively washed and incubated with Alexa-488- or Alexa-555-labeled secondary antibodies for 1 h at room temperature. The slides were mounted and subjected to examinations by using sequentially scanning on a laser scanning confocal microscope (Fluoview FV1000, Olympus, Japan), with photos being taken, and the co-localization analyzed by the Image Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD, USA). The summarized co-localization efficiency data was expressed as Pearson correlation coefficient (PCC) as described previously [16 (link)].
5
Western Blot Analysis of Vascular Markers
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Western blot analysis was performed as described previously [6 (link)]. In brief, proteins from the CAMs or dissected coronary arteries were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins of these samples were then electrophoretically transferred onto a PVDF membrane (Millipore, USA). The membrane was blocked with 5% nonfat milk and then probed with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase-labeled IgG (1:5000). Primary antibodies used were mouse anti-vimentin (1:1000, Abcam), rabbit anti-α-smooth muscle actin (SMA, 1:1000, Abcam), rabbit anti-calponin (1:1000, Abcam), rabbit anti-p62 (1:200, Abcam), mouse anti-ubiquitin (1:1000, Cell Signaling), rabbit anti-CDK1 (1:1000, Cell Signaling) or goat anti-β-actin (1:2000, Santa Cruz). The immuno-reactive bands were detected by chemiluminescence methods and visualized on Kodak Omat X-ray films. Densitometric analysis of the images obtained from X-ray films was performed using the Image J software (NIH).
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