ab3085, by Abcam - Product details

1

Western Blot Quantification of CCND2

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Equal amounts of protein were heated to 70°C for 10 mins with NuPAGE® LDS Sample Buffer (4X) (Life Technologies) and run on NuPAGE® Novex® 4-12% Bis-Tris gels (Life Technologies) and transferred to PVDF membranes (Life Technologies). Membranes were blocked for one hour at room temperature (RT) with 5% dried skimmed milk (Marvel) in PBS with 0.1% Tween-20. Membranes were then incubated with primary antibody at a dilution of 1:200 for CCND2 (ab3085, Abcam, Cambridge, UK) and 1:10,000 for β-actin (ab8226, Abcam) overnight in blocking solution at 4°C with slight agitation. The membranes were washed with 0.1% Tween-20 in PBS and then incubated with secondary antibody for 1 hour at RT. The secondary antibody (polyclonal goat anti-mouse immunoglobulins/HRP, P0447, Dako, Ely UK) was diluted 1:10,000 in blocking solution. Membranes were then washed in PBS with 0.1% Tween-20, developed with SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) and visualized using a Geldoc XR system (Biorad, Hemel Hempstead, UK). Experiments were performed in triplicate and .tiff images were analyzed using ImageJ^{24 (link)}. Bands were quantified and intensities determined relative to the corresponding ‘0’ time point. CCND2 intensity was normalized with β-actin.

Mirzaa G., Parry D.A., Fry A.E., Giamanco K.A., Schwartzentruber J., Vanstone M., Logan C.V., Roberts N., Johnson C.A., Singh S., Kholmanskikh S.S., Adams C., Hodge R.D., Hevner R.F., Bonthron D.T., Braun K.P., Faivre L., Rivière J.B., St-Onge J., Gripp K.W., Mancini G.M., Pang K., Sweeney E., van Esch H., Verbeek N., Wieczorek D., Steinraths M., Majewski J., Boycot K.M., Pilz D.T., Ross M.E., Dobyns W.B, & Sheridan E.G. (2014). De novo CCND2 mutations leading to stabilization of cyclin D2 cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome. Nature genetics, 46(5), 510-515.

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2

Protein Expression in P4 Hearts

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Hearts of P4 pups were homogenized and protein isolated using the RIPA lysis buffer system (Santa Cruz Biotechnology). Protein concentrations were quantified using the BCA protein assay (ThermoScientific) and all samples were loaded with equal protein onto 10% polyacrylamide gel with 0.1% sodium dodecyl sulfate (SDS). Proteins were then separated by electrophoresis and transferred onto nitrocellulose membranes. Non-specific binding sites were blocked with Tris-buffered saline solution (TBS) containing 5% dry milk. The membranes were incubated with primary antibodies against cyclin D2 (ab3085, Abcam; 1:1000), and p27 (ab7961, Abcam; 1:1000). After washing, membranes were incubated with secondary antibodies. Proteins were visualized with enhanced chemiluminescence reagents and Western blots were exposed to Hyperfilm. To assure equal loading and minimize variability among gels, samples were normalized to GAPDH.

Gay M.S., Li Y., Xiong F., Lin T, & Zhang L. (2015). Dexamethasone Treatment of Newborn Rats Decreases Cardiomyocyte Endowment in the Developing Heart through Epigenetic Modifications. PLoS ONE, 10(4), e0125033.

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3

Cardiac Protein Abundance in Anoxia

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HIF-1α, ET_A-receptor (ET_AR), and ET_B-receptor (ET_BR) protein abundance in the P4 heart was measured from control and anoxia groups. The protein abundance of cyclin D2 and p27 was measured in P4 hearts from control and anoxia groups as well as in the presence and absence of PD156707. Tissues were homogenized and protein isolated using the RIPA lysis buffer system (Santa Cruz Biotechnology). Protein concentrations were quantified using the BCA protein assay (ThermoScientific) and all samples were loaded with equal protein onto 7.5% (HIF-1α) or 10% (ET_AR, and ET_BR, cyclin D2, and p27) polyacrylamide gel with 0.1% sodium dodecyl sulfate (SDS). Proteins were then separated by electrophoresis and transferred onto nitrocellulose membranes. Non-specific binding sites were blocked with Tris-buffered saline solution (TBS) containing 5% dry milk. The membranes were incubated with primary antibodies against HIF-1α (sc10790, Santa Cruz Biotechnology; 1:500), ET_AR (sc33536, Santa Cruz Biotechnology; 1:500), ET_BR (sc33538, Santa Cruz Biotechnology; 1:500), cyclin D2 (ab3085, Abcam; 1:1000), and p27 (ab7961, Abcam; 1:1000). After washing, membranes were incubated with secondary antibodies. Proteins were visualized with enhanced chemiluminescence reagents and western blots were exposed to Hyper film. Kodak image software was used to quantify all results.

Paradis A.N., Gay M.S., Wilson C.G, & Zhang L. (2015). Newborn Hypoxia/Anoxia Inhibits Cardiomyocyte Proliferation and Decreases Cardiomyocyte Endowment in the Developing Heart: Role of Endothelin-1. PLoS ONE, 10(2), e0116600.

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4

Renal Protein Expression Analysis

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Proteins were extracted from the whole kidney and homogenized in protein lysis buffer. Equal amount of protein (60 μg) was subjected to SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. The blots were blocked at room temperature (RT) for 1h with 5% nonfat dry milk in Tris-buffered saline (TBS) and probed with the following antibodies: rabbit anti-NHE3 (Catalog No.: AB3085, Abcam, Cambridge, MA), anti-NKCC2 (Catalog No.: SPC-401, Stressmarq Biosciences Inc., Canada), anti-NCC (Catalog No.: SPC-402, Stressmarq Biosciences Inc., Canada), anti-ENaCα (Catalog No.: SPC-403, Stressmarq Biosciences Inc., Canada) and anti-ENaCγ (Catalog No.: SPC-405, Stressmarq Biosciences Inc., Canada) at a dilution of 1: 1,000 at 4°C overnight. After incubated with secondary antibodies (Catalog No.: sc-2004, goat anti-rabbit IgG, Santa Cruz) at RT for 1h, membranes were visualized using Pierce™ ECL Western Blotting Substrate (Thermo Scientific). Quantification was performed using Image-Pro Plus 6.0 software.

Liu M., Zhu Y., Sun Y., Wen Z., Huang S., Ding G., Zhang A., Jia Z, & Zhang Y. (2017). MnTBAP therapy attenuates the downregulation of sodium transporters in obstructive kidney disease. Oncotarget, 9(1), 394-403.

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5

Quantification of Cardiac Proliferation Markers

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VSD heart tissue sections (8μm) were used for Ki67 and cyclin D2 staining. The initial slide was selected using a random number generator and every seventh slide after was chosen for microscopy by systematic random selection. The slides were stained overnight with mouse monoclonal antibodies against cardiac troponin T (Abcam, ab8295, 1:200) and an Alexa Fluor® 488-conjugated rabbit anti-Ki67 antibodies (Abcam, ab154201; 1:200) or rabbit antibody against cardiac sarcomeric alpha actinin (Abcam, ab90776, 1:200) and mouse antibody against cyclin D2(Abcam, ab3085,1:200). After three washes, the sections were incubated with Alexa Fluor® 555-conjugated anti-mouse secondary antibodies (Abcam, ab150107; 1:200) or Alexa Fluor® 555-conjugated anti-rabbit secondary antibody (CST, 4409, 1:500) and an Alexa Fluor® 488-conjugated anti-mouse secondary antibody (Abcam, ab150073, 1:500) for 30 min. Three researchers who were blinded to sample identities performed the quantification of cellular Ki67 and cyclin D2 events using either manual counting or digital thresholding (image segmentation and the creation of a binary image from a gray scale) methods. Analysis of the converted binary images was performed using ImageJ (National Institutes of Health).

Huang Y., Hong H., Li M., Liu J., Jiang C., Zhang H., Ye L, & Zheng J. (2017). Age-Dependent Oxidative DNA Damage Does Not Correlate with Reduced Proliferation of Cardiomyocytes in Humans. PLoS ONE, 12(1), e0170351.

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6

Western Blot Quantification of CCND2

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Equal amounts of protein were heated to 70°C for 10 mins with NuPAGE® LDS Sample Buffer (4X) (Life Technologies) and run on NuPAGE® Novex® 4-12% Bis-Tris gels (Life Technologies) and transferred to PVDF membranes (Life Technologies). Membranes were blocked for one hour at room temperature (RT) with 5% dried skimmed milk (Marvel) in PBS with 0.1% Tween-20. Membranes were then incubated with primary antibody at a dilution of 1:200 for CCND2 (ab3085, Abcam, Cambridge, UK) and 1:10,000 for β-actin (ab8226, Abcam) overnight in blocking solution at 4°C with slight agitation. The membranes were washed with 0.1% Tween-20 in PBS and then incubated with secondary antibody for 1 hour at RT. The secondary antibody (polyclonal goat anti-mouse immunoglobulins/HRP, P0447, Dako, Ely UK) was diluted 1:10,000 in blocking solution. Membranes were then washed in PBS with 0.1% Tween-20, developed with SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific) and visualized using a Geldoc XR system (Biorad, Hemel Hempstead, UK). Experiments were performed in triplicate and .tiff images were analyzed using ImageJ^{24 (link)}. Bands were quantified and intensities determined relative to the corresponding ‘0’ time point. CCND2 intensity was normalized with β-actin.

Mirzaa G., Parry D.A., Fry A.E., Giamanco K.A., Schwartzentruber J., Vanstone M., Logan C.V., Roberts N., Johnson C.A., Singh S., Kholmanskikh S.S., Adams C., Hodge R.D., Hevner R.F., Bonthron D.T., Braun K.P., Faivre L., Rivière J.B., St-Onge J., Gripp K.W., Mancini G.M., Pang K., Sweeney E., van Esch H., Verbeek N., Wieczorek D., Steinraths M., Majewski J., Boycot K.M., Pilz D.T., Ross M.E., Dobyns W.B, & Sheridan E.G. (2014). De novo CCND2 mutations leading to stabilization of cyclin D2 cause megalencephaly-polymicrogyria-polydactyly-hydrocephalus syndrome. Nature genetics, 46(5), 510-515.

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Ab3085

Lab products found in correlation

6 protocols using ab3085

Western Blot Quantification of CCND2

Protein Expression in P4 Hearts

Cardiac Protein Abundance in Anoxia

Renal Protein Expression Analysis

Quantification of Cardiac Proliferation Markers

Western Blot Quantification of CCND2

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