Protein samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). Membranes were blocked with fat‐free milk combined with tris‐buffered saline plus tween 20 for one hour at room temperature and then incubated with the appropriate primary antibody and horseradish peroxidase conjugated secondary antibodies. Imager was used to visualize the blots. The primary antibodies used in this study were as follows: anti‐CDK4 (#12790, 1:1000, cell signaling), Anti‐Cyclin D1 antibody (#2978, 1:1000, cell signaling), Anti‐phospho‐EGFR antibody (#47724, 1:1000, cell signaling), Anti‐Phospho‐RB (ser780, #9307, 1:1000, cell signaling), anti‐E2F1 (#666515,1:1000,Proteintech), CDKN2a (#80772, 1:1000, cell signaling); and β‐actin (ab92552, 1:1000).
Anti cyclin d1 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Anti-cyclin D1 antibody is a laboratory reagent used to detect and analyze the presence and levels of cyclin D1 protein in biological samples. Cyclin D1 is a key regulator of cell cycle progression and is involved in the control of cell proliferation. The antibody can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and flow cytometry, to study the expression and localization of cyclin D1 in different cell types and tissues.
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Lab products found in correlation
Anti β actin antibody, by Cell Signaling Technology (4 mentions)
Anti cdk4 antibody, by Cell Signaling Technology (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Piercetm bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Anti akt1 antibody, by Cell Signaling Technology (2 mentions)
Anti β catenin antibody, by Cell Signaling Technology (2 mentions)
Dc protein assay kit, by Bio-Rad (2 mentions)
Anti cdk6 antibody, by Cell Signaling Technology (2 mentions)
Anti cdk4, by Cell Signaling Technology (1 mentions)
Anti phospho egfr antibody, by Cell Signaling Technology (1 mentions)
Anti e2f1, by Proteintech (1 mentions)
Anti phospho rb, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti bax antibody, by Cell Signaling Technology (1 mentions)
Axio vert a1 inverted fluorescent microscope, by Zeiss (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Anti p erk1 2 antibody, by Cell Signaling Technology (1 mentions)
Envision hrp secondary antibody, by Agilent Technologies (1 mentions)
Enhanced chemiluminescence reagent, by GE Healthcare (1 mentions)
Anti ps6 antibody, by Cell Signaling Technology (1 mentions)
22 protocols using anti cyclin d1 antibody
1
Western Blot Analysis of Cell Cycle Regulators
2
Immunofluorescence and Immunohistochemistry Assays for Breast Cancer
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For IF assay, breast cancer cells were cultured to ~70% confluence, and then fixed with 4% paraformaldehyde for 30 min at room temperature. After washing with PBS, cells were permeabilized with 0.5% Triton X- 100 in PBS for 20 min on ice and then blocked with blocking buffer (1× PBS, 5% BSA, 0.3% Triton X-100) for 60 min at room temperature. Samples were incubated with anti-Akt1 antibody (1:200; Cell Signaling Technology, USA) for overnight at 4 °C. After wash with PBS, samples were incubated with Alexa Fluor 488 conjugated goat rabbit anti-IgG secondary antibody (1:250; Cell Signaling Technology, USA) for 1 h at room temperature in the dark. Finally, all samples were counterstained with Hoechst 33342 (1:1000 dilution; Invitrogen, USA) and observed by Zeiss Axio Vert. A1 inverted fluorescent microscope (Carl Zeiss Microscopy GmbH, Germany). For IHC assay, paraffin sections of tumors were incubated with primary antibodies: anti-Akt1 antibody (1:100), anti-β-catenin antibody (1:100), anti-cyclin D1 antibody (1:100), and anti-Bax antibody (1:100) (Cell Signaling Technology, USA) at 4 °C overnight and then incubated with the GTVision III Detection System/Mo&Rb Kit (Gene Tech Co., Ltd., Shanghai, PR China).
3
Immunohistochemical Analysis of TEM8, Cyclin D1, and pERK1/2
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Four-μm-thick sections were deparaffinized, rehydrated in serially graded ethanol, heated in citric buffer (pH 6.0) once for 20 min in a microwave oven for antigen retrieval, and blocked with 3% hydrogen peroxide for 15 min. The samples were then labeled with anti-TEM8 antibody (1:300, Abcam, #21270), anti-cyclin D1 antibody (1:200, cell signaling technology) and anti-pERK1/2 antibody (1:200, cell signaling technology) at 4 °C overnight. The next day, after washing with phosphate-buffered saline (PBS), the sections were incubated with EnVision-HRP secondary antibody (DAKO, Carpinteria, CA) for 30 min at 37 °C in a water bath, washed with PBS, stained with 0.5% diaminobenzidine and counterstained with Mayer’s hematoxylin, then air dried, and mounted with resinene.
4
Comprehensive Western Blot Analysis
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Western blotting analysis was performed as previously described (22 (link)). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE). After transferring onto membranes, the proteins were probed with corresponding antibodies and detected with ECL detection reagents (GE Healthcare). The primary antibodies for anti-caspase-3 antibody, anti-cleaved caspase-3 antibody, anti-poly-ADP-ribose polymerase (PARP) antibody, anti-phosphorylated mTOR (p-mTOR) antibody, anti-p-p70S6K antibody, anti-p-S6 antibody, anti-p-4E-BP1 antibody, anti-CDK4 antibody, anti-Cyclin D1 antibody, anti-p-Rb antibody, and anti-Rb were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin was from Sigma (St. Louis, MO, USA). Secondary anti-mouse and rabbit antibodies were purchased from Thermo Fisher Scientific, Inc. (Waltham, MA, USA), polyvinylidene difluoride membranes were from Millipore (Billerica, MA, USA), and enhanced chemiluminescence reagents were from GE Healthcare (Buckinghamshire, UK).
5
Protein Extraction and Western Blotting
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Total protein was extracted from cells or tissues using RIPA buffer and the concentrations were measured by using Bio-Rad protein assay reagent (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's instructions. For western blotting, protein samples (25 µg) was subjected to a 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transferred onto a polyvinylidene fluoride (PVDF) membrane (GE Healthcare Life Sciences, Little Chalfont, UK). Subsequently, the PVDF membrane was blocked in 5% nonfat milk in 0.1% Tris-buffered saline (TBS)-Tween (TBST) for 1 h at room temperature. Thereafter, the membrane was probed with the anti-IGF-1 antibody (ab40789; Abcam, Cambridge, MA, USA), anti-cyclin D1 antibody (#2922; Cell Signaling Technology, Inc., Danvers, MA, USA), or anti-CDK1 (ab18; Abcam) overnight at 4°C. Following this, membranes were incubated with horseradish-peroxidase secondary antibody (Cell Signaling Technology, Inc.) at room temperature for 2 h. Subsequent to being washed 3 times with TBST, the blotted proteins were visualized with enhanced chemiluminescence detection system (EM Millipore, Billerica, MA, USA). GAPDH served as the internal control.
6
Western Blot Analysis of Cyclin-D1
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Proteins were extracted from frozen tissue using PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Seongnam, Korea) on ice, according to the manufacturer’s instructions. Protein concentrations were measured using a DC protein assay kit (Bio-Rad, Hercules, CA, USA). Afterwards, 20 µg of protein extracted from each line was separated using sodium dodecyl sulfate gel electrophoresis, and the separated proteins were transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Membranes were blocked with 5% skim milk in Tris-buffered saline for 1 h and incubated with an anti-cyclin-D1 antibody (1:1000; Cell Signaling Technology, Beverly, Massachusetts, USA) at 4 °C overnight. Following incubation, the membranes were washed and incubated with a goat anti-rabbit horseradish peroxidase-labelled secondary antibody (1:5000; Bethyl Laboratories, Montgomery, TX, USA) for 1 h. Immunoreactive bands were normalized to β-actin (1:1000; Santa Cruz Biotechnology) as a loading control and visualized using ImageQuant Las 4000 mini (GE Healthcare Life Sciences, Chicago, IL, USA). Band densities were analysed using the ImageJ software (ImageJ 1.43u, National Institutes of Health, USA).
7
Immunohistochemical Analysis of FXR and Cyclin D1 in TMAs
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IHC staining for FXR and cyclin D1 expression in TMAs was performed as described4 (link). Anti-bile acid receptor (NR1H4) antibody (Abcam, Cambridge, MA) and anti-cyclin D1 antibody (Cell Signaling Technology, Beverly, MA) were applied at 1:100 and 1:50, respectively. Concentration-matched nonspecific rabbit IgG was used as a isotype control. Two trained pathologists viewed the IHC staining results and reached a final consensus. The scoring for the staining intensity was as follows: negative (0), weak (1), moderate (2) and intense (3). The scoring for the percentage of positive cells was as follows: 0% (0), 1–25% (1), 26–50% (2), 51–75% (3) and 76–100% (4). The final FXR or cyclin D1 IHC score was obtained by multiplying the intensity and percentage scores, which were defined as low (including scores of 0–4) or high (6–12) expression.
8
Western Blot Analysis of Cell Cycle Regulators
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Western blot analysis was performed according to previously described procedures16 . We used primary antibodies, including anti-bile acid receoptor (NR1H4) antibody, anti-SHP antibody (Santa Cruz, CA), anti-phospho-Rb antibody, anti-cyclin D1 antibody, anti-cyclin E1 antibody, anti-CDK2 antibody, anti-CDK4 antibody, anti-CDK6 antibody, anti-p21Waf1/Cip1 antibody, anti-p27Kip1 antibody, and anti-β-actin antibody (Cell Signaling Technology) according to the manufacturer’s recommended dilutions.
9
Analyzing ccRCC Cell Signaling Pathways
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ccRCC cells were lysed with NP-40 lysis buffer according to the manufacturer’s instructions. Then, the protein concentration of each sample was measured using PierceTM BCA protein assay kit (Thermo, MA, USA). Equal amounts of cell lysates were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. After being blocked in 5% fat-free milk, the PVDF membranes were incubated with primary antibodies overnight at 4 °C. The following antibodies were used: anti-HIF2α antibody (Catalog number: ab199, 1:1000) was purchased from abcam (Cambridge, UK); anti-E2F1 antibody (Catalog number: 3742, 1:1000), anti-Cyclin D1 antibody (Catalog number: 55506, 1:1000) and anti-Beta-actin antibody (Catalog number: 4970, 1:1000) were all purchased from Cell signaling Technology (MA, USA); anti-VEGF antibody (Catalog number: 19003-1-AP, 1:1000) was purchased from Proteintech (Wuhan, China). Then membranes were incubated with HRP-conjugated anti-rabbit IgG at room temperature for 1 h and signal detection was visualized using a western blot substrate kit (Tanon, Shanghai, China).
10
Cell Cycle Arrest Protein Analysis
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Proteins were extracted from TCs using the pro‐prep protein extraction solution (iNtRON Biotechnology). The protein concentrations were determined using the DC protein assay kit (Bio‐Rad, USA). Approximately, 10 µL of each protein was separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to a polyvinylidene difluoride membrane (Millipore, USA). The membranes were blocked with a blocking buffer containing 5% skim milk in Tris‐buffered saline. Cell cycle arrest was detected using anti-procaspase-3 (dilution, 1:1,000; Santa Cruz Biotechnology) and anti-cyclin D1 antibody (dilution, 1:1,500; Cell Signalling Technology, USA). An anti‐β‐actin antibody (dilution, 1:1,000; Santa Cruz Biotechnology) was used as a loading control. Immunoreactive bands were detected by chemiluminescence (Advansta, USA).
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