40,000 cells/well were seeded in Labtek slides, followed by fixation in Formalin after overnight incubation. After fixing, the background produced from formalin was quenched with NH4Cl for 15 min and then samples were blocked and permeabilized with 0.5% Goat serum + Triton-X 0.3% in PBS for 2 h, prior to overnight incubation with primary antibodies: (p-CREB: 1:1000 (Cell Signaling 9198), PNCK 1:250 (Invitrogen PA5-99601) Angiopoietin 1 (Abcam 133425) 2.5μ/mL, Angiopoietin 2 (Abcam 153934) 1:500, Cyclin D1 (Abcam 16663) 1:150, p21 (Abcam 109520) 1:100, p27 (Cell Signaling 3886) 1:800, Cyclin A (Cell Signaling 67955) 1:500, Cyclin B2 (Abcam 185622) 1:300, Ki67 (Abcam 15580) 0.5μ/mL. Alexa Fluor 555 conjugated secondary antibodies (1:350) were used to visualized cells under fluorescent microscopy for overexpression experiments. Alexa Fluor 488 was used in dsiRNA knockdown experiments.
Cyclin b2
Manufactured by Abcam
Cyclin B2 is a regulatory protein that plays a crucial role in the progression of the cell cycle. It is a member of the cyclin family, which are essential for the activation of cyclin-dependent kinases (CDKs) that drive the cell cycle. Cyclin B2 specifically activates CDK1, which is involved in the transition from the G2 phase to the M phase of the cell cycle, promoting mitosis.
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Lab products found in correlation
4 protocols using cyclin b2
1
Immunofluorescence Staining of Signaling Proteins
2
Comprehensive Immunohistochemical Staining Protocol
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A standard hematoxylin and eosin staining procedure was performed in all samples17 (link)-20 (link). Formalin-fixed, paraffin-embedded tissue samples were sliced consecutively at 4 μm. tissues were mounted on polylysine-coated glass slides. Endogenous peroxidase of deparaffinized sections was blocked through incubation with 3% hydro- gen peroxide for 15 min. The samples were then deparaffinized, with gradient rehydration in ethanol. The following antibodies were used for IHC staining: CKAP4 (Abcam), Cyclin B1 (Abcam), and Cyclin B2 (Abcam). Specific dilution of each enzyme was per manufacturer's protocol. We used DAB (diaminobenzidine tetrahydrochloride) solution for color developing and all slides were finalized by counterstain with Mayer's hematoxylin blue. Positive and negative controls for all enzyme labelers were referenced using upon the Human Protein Atlas platform. Quantification was performed in the method aforementioned.
3
Cell Cycle Regulation in Cancer
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p53 (#sc-126), normal mouse IgG and HRP (#sc-2025; #sc-2748) and ERα (#sc-543) were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. MGMT (#MAB16200), ki-67 (#MAB4190), AURKB (#MAB 04-1036) and TOP2A (#MAB 4197MI) antibodies were purchased from Chemicon-Millipore, Billerica, MA. CDC2 (#9112S), phospho-CDC2 (Try15) (#9111S), phospho-CDC2 (Tyr14) (#2543S), CDC20 (#4823S), phospho-CDC20 (ser51) (#8038), p-AURKB (#2914S), Cyclin A2 (#4656S), Cyclin D1(#2922), survivin (#2803), p21 (#2946), cleavedPARP (#9546), cytochrome C (#4272) and PUMA (#4976) were purchased from Cell Signaling, Beverly, MA. Phospho-TOP2A (p-s1106) (#ab75765), KIF20A (#ab70791), phospho-KIF20A (p-s528) (#ab63547) and Cyclin B2 (#ab82287) were purchased from Abcam, Cambridge, MA. β-Actin (#A2066) antibody was purchased from Sigma-Aldrich, St. Louis, MO. O6 benzyl guanine (BG; MGMT Blocker) was purchased from Sigma-Aldrich (St. Louis, MO). Temozolomide was obtained from Aurora Saint Luke’s Pharmacy. BG and TMZ were dissolved in DMSO and subsequent dilutions were made with tissue culture media. Equal amounts of DMSO were used in tissue culture media for untreated controls.
4
Quantitative Analysis of CKAP4 Expression
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For quantitative PCR, a standard protocol was followed. Total RNA was extracted with Trizol and was converted to cDNA. Primers for CKAP4 was 5'- CCG TGG AAT CAC TCC AGA AGG -3' as forward, and 5'-AGT CCT GAG CAT TTT CAA GTC C-3' as reverse. GAPDH was used as internal reference. cDNA was subject to the ABI 7500 for quantitative PCR procedure and the program was per manufacturer's instruction of SYBR Green system. The expression of CKAP4 was calculated according to internal references and were expressed as folds over the control group.
For western blotting, total protein of cell lysates was extracted and equal protein amount of 25 μg was loaded onto 10% sodium dodecyl sulphate polyacrylamide gel. Gels were subsequently transferred to nitrocellulose membrane. The membranes were blocked with 5% non-fat milk. Primary anti- human CKAP4 (Abcam), Cyclin B1 (Abcam), Cyclin B2 (Abcam), CDK1 (Abcam), and Actin (Abcam) antibodies were then added at the dose recommended by the manufacturers and membranes were kept at 4°C overnight. Procedure was finalized by enhanced chemiluminescence.
For western blotting, total protein of cell lysates was extracted and equal protein amount of 25 μg was loaded onto 10% sodium dodecyl sulphate polyacrylamide gel. Gels were subsequently transferred to nitrocellulose membrane. The membranes were blocked with 5% non-fat milk. Primary anti- human CKAP4 (Abcam), Cyclin B1 (Abcam), Cyclin B2 (Abcam), CDK1 (Abcam), and Actin (Abcam) antibodies were then added at the dose recommended by the manufacturers and membranes were kept at 4°C overnight. Procedure was finalized by enhanced chemiluminescence.
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