MA5-14512

antibody from Invitrogen Antibodies

Targeting: CCND1 BCL1, D11S287E, PRAD1, U21B31

Provider product page for MA5-14512

Western blot

Immunocytochemistry

Immunohistochemistry

Flow cytometry

Other assay

Antibody data

Antibody Data
Antigen structure
References [301]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [5]
Immunohistochemistry [5]
Other assay [8]

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Product number: MA5-14512 - Provider product page
Provider: Invitrogen Antibodies
Product name: Cyclin D1 Recombinant Rabbit Monoclonal Antibody (SP4)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: MA5-14512 targets Cyclin D1/Bcl-1 in IHC (P), ICC/IF and WB applications and shows reactivity with Human and Mouse samples. The MA5-14512 immunogen is a synthetic peptide from C-terminus of human cyclin D1.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Antibody clone number: SP4
Vial size: 500 µL
Concentration: 0.045 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

CCND1 protein structure - MA5-14512 shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Cyclin D1 was achieved by transfecting HeLa cells with PKM2 specific siRNAs (Silencer® select Product # S229, S230). Western blot analysis (Fig. a) was performed using whole cell extracts from the Cyclin D1 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Cyclin D1 Recombinant Rabbit Monoclonal Antibody (SP4) (Product # MA5-14512), 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Cyclin D1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Cyclin D1 Recombinant Rabbit Monoclonal Antibody (SP4) (Product # MA5-14512) and a 33 kDa band corresponding to Cyclin D1 was observed across all the cell lines and tissues tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), A549 (Lane 2), MCF-7(Lane 3), K-562 (Lane 4) and tissue extracts of Mouse Brain(Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Cyclin D1/Bcl-1 (green) showing staining in the nucleus of HeLa cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Cyclin D1/Bcl-1 monoclonal antibody (Product # MA5-14512) in 3% BSA-PBS at a dilution of 1:200 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 100x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Cyclin D1/Bcl-1 (green) showing staining in the nucleus of MCF-7 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Cyclin D1/Bcl-1 monoclonal antibody (Product # MA5-14512) in 3% BSA-PBS at a dilution of 1:200 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 100x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of Cyclin D1/Bcl-1 (green) showing staining in the nucleus of NIH-3T3 cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a Cyclin D1/Bcl-1 monoclonal antibody (Product # MA5-14512) in 3% BSA-PBS at a dilution of 1:200 and incubated overnight at 4 ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. F-actin (red) was stained with a fluorescent red phalloidin and nuclei (blue) were stained with Hoechst or DAPI. Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of Cyclin D1 was achieved by CRISPR-Cas9 genome editing using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR671278_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Immunofluorescence analysis was performed on wild type HeLa cells (panel a,b) and HeLa Cyclin D1 KO cells (panel c,d). Cells were fixed, permeabilized, and labelled with Cyclin D1 Recombinant Rabbit Monoclonal Antibody (SP4) (Product # MA5-14512, 1:200 dilution), followed by Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731TR, 1:2,000). Nuclei (blue) were stained using ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962), and Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Loss of signal (panel c,d) upon CRISPR mediated knockout (KO) confirms that antibody is specific to Cyclin D1 (green). The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of was performed using 70% confluent log phase HeLa cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 10 minutes at room temperature. The cells were labeled with Cyclin D1 Recombinant Rabbit Monoclonal Antibody (SP4) (Product # MA5-14512) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (1:2000 dilution) for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing Cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Formalin-fixed, paraffin-embedded human mantle cell lymphoma stained with Cyclin D1 using peroxidase-conjugate and DAB. Note nuclear staining of tumor cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Cyclin D1 showing staining in the nucleus of paraffin-embedded mouse liver tissue (left) compared to an isotype control (right). To expose target proteins, antigen retrieval was performed using a sodium citrate buffer (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min. at room temperature, washed with ddH2O and PBS, and probed with a Cyclin D1 monoclonal antibody (Product # MA5-14512) diluted in 3% BSA-PBS at a dilution of 1:50 overnight at 4ºC in a humidified chamber. Tissues were washed in PBST and detection was performed using a biotin-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Data provided courtesy of Antibody Resource.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Cyclin D1/Bcl-1 showing staining in the nucleus of paraffin-treated human colon carcinoma (right) compared with a negative control in the absence of primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Cyclin D1/Bcl-1 monoclonal antibody (Product # MA5-14512) diluted by 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Cyclin D1/Bcl-1 showing staining in the nucleus of paraffin-treated human tonsil tissue (right) compared with a negative control in the absence of primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Cyclin D1/Bcl-1 monoclonal antibody (Product # MA5-14512) diluted by 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Cyclin D1/Bcl-1 showing staining in the nucleus of paraffin-treated mouse colon tissue (right) compared with a negative control in the absence of primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Cyclin D1/Bcl-1 monoclonal antibody (Product # MA5-14512) diluted by 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 The expression of D-cyclins in mantle cell lymphoma The differential expression of D-cyclin staining in two case examples of mantle cell lymphoma is shown. Case 1 shows intense cyclin D1 and weak cyclin D2 staining. Case 2 shows weak cyclin D1 and strong cyclin D2 staining. Both cases lack staining for cyclin D3.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. Overexpression of miR-532-5p downregulates CCND1 expression in HG-induced cells. (A) Targeted binding sites of miR-532-5p and CCND1. (B) Luciferase reporter gene assay was performed to verify the binding between miR-532-5p and CCND1. The expression of CCND1 was determined using (C) reverse transcription-quantitative PCR and (D) western blotting. **P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6. Overexpression of miR-532-5p improves the impaired functions of secreted insulin in HG-induced cells by downregulating CCND1. (A) RT-qPCR and (B) western blotting were performed to detect the expression of CCND1. (C) ELISA assay was used to detect the levels of insulin secretion. RT-qPCR was conducted to detect the expression levels of (D) Insulin1 and (E) Insulin2. **P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Lamin B1 build up inhibits cell cycle progression and proliferation: The expression of proteins involved in cell cycle progression and apoptosis was evaluated in U87-MG cells 96 h after transduction. Lamin B1 overexpressing cells (ovLMNB1) were compared to wild type (WT) and mock-transduced (GFP) samples. ( A ): Western blot results representative of five independent experiments. The expression of the denoted proteins was assayed. Samples were normalized by performing total protein normalization vs. wild type cells. ( B ): Quantitative analysis of protein expression, wild type cells (WT) were used as reference sample. ** denotes p < 0.01 vs. corresponding mock-transduced sample (GFP). ( C ): Immunofluorescence staining of Ki67 at 63x magnification (bar: 10 uM) in wild-type (WT), mock-transduced (GFP), and Lamin B1-overexpressing (ovLMNB1) cells. Nuclei were stained using Hoechst 33342 (blue). The staining of Ki67 is marked in WT and GFP samples, while it becomes faint in cells overexpressing Lamin B1. Results are representative of at least five different fields.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. Garcinol regulates the expression of cell cycle-related proteins in EC cells. Effect of garcinol on the expression of cell cycle-regulated genes in EC cells were detected by western blotting. ISH and HEC-1B cells were cultured for 24 h and then treated with and without garcinol (0, 1, 5, 10 and 20 uM) for another 24 h. (A) Total protein was collected, and western blot analyses were performed for p53, p21, cyclin D1, cyclin B1, CDK2, CDK4. beta-actin was used as alpha loading control. (B) Band density was analyzed by ImageJ, and the results are expressed as the mean +- SD, n=3. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 TWIST1 enhanced Wnt/beta-catenin signaling and regulated expression of vascularization-related genes A: Luciferase analysis showed that Wnt/beta-catenin signaling activity was increased in HEK293STF cells transfected with TWIST1 plasmids. B, C: Western blotting showed that cyclin D1 and phosphorylation of GSK3beta proteins increased when TWIST1 was overexpressed in HRECs. Western blotting with anti-Flag and TWIST1 showed successful transfection of TWIST1 plasmids. GAPDH was used as the loading control. Data are presented as mean+-standard error of the mean ( SEM ). Sample size n =4. Data are from one typical experiment of three independent experiments. ** : P< 0.01; * : P< 0.05, two-tailed t -test. D: RNA-seq showed that vascularization-related genes were up-regulated when TWIST1 was overexpressed in HRECs. E: RT-qPCR verified RNA-seq results in HRECs for indicated genes. *** : P< 0.001. Sample size n =4. F: RT-qPCR verified that expression of most of TWIST1-targeting genes identified by RNA-seq were increased in the lungs of Tg-Twist1 iEC+ mice. *** : P< 0.001; ** : P< 0.01; * : P< 0.05; ns, no significant difference. Sample size n =4.