HEK 293T, HCT116, HT29, LoVo, SW480, SW620 and RKO cells were obtained from our collaborators and maintained in DMEM (GIBCO) supplemented with 10% FBS (HyClone). CSBF/C10orf99 was synthesized in Chinese Peptide Company (Hangzhou, China) with a purity > 97%. Gal1 was purchased from R&D Systems (1152-GA-050). Antibodies used in this paper were: mouse anti-myc (9E10) (# SAB4700447, Sigma-Aldrich), rabbit anti-HA (C29F4) (# 3724, Cell Signaling Technology), goat anti-Human IgG (Fc specific) (# I2136, Sigma-Aldrich), anti-cyclin D1 (# 2926, Cell Signaling Technology), anti-cyclin D3 (# 2936, Cell Signaling Technology), anti-CDK4 (# 2906, Cell Signaling Technology), anti-CDK6 (# 3136, Cell Signaling Technology), anti-phospho-Cyclin B1 (Ser147) (# 4131, Cell Signaling Technology), anti-SUSD2 rabbit polyclonal antibody (# HPA004117, Sigma-Aldrich), anti-β-tublin (# T8328, Sigma-Aldrich) and anti-GAPDH (# 2118, Cell Signaling Technology). Primary cancer tissues and paired adjacent tissues were obtained from patients under primary surgery at Peking University Cancer Hospital (Beijing, China), with patients' consent and institutional ethics approval. Fresh human tissues were fixed with 10% formalin in PBS for immunohistochemistry, or frozen in liquid nitrogen for RNA extraction. This investigation was carried out after approval by the Ethics Committee of Peking University Cancer Hospital.
Anti cyclin d3
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-Cyclin D3 is a primary antibody that specifically binds to Cyclin D3, a regulatory protein involved in the cell cycle. It is designed for use in various research applications, including Western blotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti cyclin d1, by Cell Signaling Technology (7 mentions)
Anti cdk4, by Cell Signaling Technology (5 mentions)
Anti cdk6, by Cell Signaling Technology (4 mentions)
Anti β actin, by Cell Signaling Technology (3 mentions)
Anti akt, by Cell Signaling Technology (3 mentions)
Image lab software, by Bio-Rad (2 mentions)
Anti n cadherin, by Cell Signaling Technology (2 mentions)
Anti mmp2, by Cell Signaling Technology (2 mentions)
Mouse anti myc 9e10, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Gal 1, by R&D Systems (1 mentions)
Rabbit anti ha c29f4, by Cell Signaling Technology (1 mentions)
Goat anti human igg fc specific, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Merck Group (1 mentions)
Anti p53, by Cell Signaling Technology (1 mentions)
Anti β actin, by Beyotime (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Anti β catenin, by Santa Cruz Biotechnology (1 mentions)
12 protocols using anti cyclin d3
1
Cell Lines and Reagents for Cancer Research
2
Protein Expression Analysis of Cell Lines Exposed to MWCNTs
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Total protein of cell line lysate (including control and 0.5 μg/mL of MWCNTs groups) was collected, and then separated on SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The antibodies used were anti-CDK4 (Cell Signaling Technology, 1:1000, Danvers, MA, USA), anti-Cyclin D1 (Cell Signaling Technology, 1:1000, Danvers, MA, USA), anti-Cyclin D3 (Cell Signaling Technology, 1:1000, Danvers, MA, USA), anti-P53 (Cell Signaling Technology, 1:1000, Danvers, MA, USA), anti-β-actin (Beyotime, 1:1000, China). The immune complexes were detected by enhanced chemiluminescence (Millipore, Billerica, MA, USA). Blots were quantified by densitometry and normalized by use of β-actin to correct for differences in loading of the proteins. For densitometric analyses, the band was quantified using Image Lab software (BioRad laboratories, Hercules, CA, USA). Each experiment was performed at least twice.
3
Regulation of SRC-3 by shRNA
4
Compound K Inhibits Cancer Stemness
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Compound K was obtained from Ilhwa Co., Ltd., (Guri, Korea) and dissolved in dimethyl sulfoxide (DMSO) at a concentration of 50 mM as a stock solution. Gelatin and Matrigel were purchased from Sigma-Aldrich (St. Louis, MO, USA) and BD Biosciences (San Jose, CA, USA), respectively. Anti-MMP-2 (64 kDa), anti-MMP-9 (84 kDa), anti-cyclin D1 (36 kDa), anti-cyclin D3 (31 kDa), anti-PARP (89, 116 kDa), anti-cleaved caspase-3 (Asp175, 17, 19 kDa), anti-cleaved caspase-9 (Asp330, 37 kDa), anti-phospho-PI3K [p85 (Tyr458)/p55 (Tyr199), 60 and 85 kDa], anti-PI3K (p85, 85 kDa), anti-phospho-Akt (Ser473, 60 kDa), anti-Akt (60 kDa), anti-phospho-mTOR (Ser2448, 289 kDa), anti-mTOR (289 kDa), anti-Nanog (42 kDa), anti-Sox2 (35 kDa), anti-Oct4 (45 kDa) and anti-β-actin (45 kDa) antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-CD133 (133 kDa) antibody was obtained from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany).
5
RANKL-Induced Osteoclast Differentiation
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We are very grateful to Dr. S.L. Teitelbaum (Washington University, St. Louis, MO, USA), and T. Kitamura (Tokyo University, Tokyo, Japan) for generously providing GST-RANKL vector and PLAT-E cells, respectively. Recombinant mouse RANKL was purchased from R&D Systems International (Minneapolis, MN, USA). Antibodies were from the following companies: anti-ATF3 and anti-β-Actin were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-Cyclin D1, anti-Cyclin D3 and anti-CDK4 were from Cell Signaling Technology (Danvers, MA, USA). THUNDERBIRD SYBR qPCR Mix was supplied by TOYOBO (Osaka, Japan). Other chemicals used were all of the highest purity commercially available.
6
Protein Expression Analysis by Western Blot
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Cell extracts were separated by SDS-PAGE. The lysate proteins were transferred from the gel to a PVDF membrane and immunoblotted with anti-Pin1 (10495-1-AP, Proteintech), anti-cyclin D1 (MAI-39546; Invitrogen), anti-cyclin D2 (#3741; Cell Signaling Technology), anti-cyclin D3 (#2936; Cell Signaling Technology), anti-CDK4 (SC-23896; Santa Cruz Biotechnology), anti-CDK6 (SC-53638; Santa Cruz Biotechnology), anti-β-catenin (610154; BD Transduction Laboratories), anti-β-actin (AbC-2002, AbClon), or anti-α/β-tubulin (#2148S; Cell Signaling Technology) antibodies. Detection was performed by chemiluminescence using Western Lightning reagent (PerkinElmer Life Sciences).
7
Gefitinib and Estradiol Effects on EGFR Signaling
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Cells were cultured in serum-free medium for 24 h and then treated with gefitinib (40 nM) or/and estradiol (20 nM) for another 8 h. Total protein was extracted from cells using cell lysis buffer (Beyotime, China) supplemented with a protease inhibitor cocktail (Roche, Germany); protein concentration was determined using the BCA protein assay (Beyotime, Beijing, China). The following primary antibodies (1:1,000) were used: anti-EGFR, anti-phospho-EGFR (Tyr1068), anti-AKT, anti-phospho-AKT (Ser473), anti-RPS6, anti-phosphor-RPS6 (Ser235/236), anti-P21, anti-CyclinD3, anti-cleaved-PARP (cPARP), and anti-beta-actin (Cell Signaling Technology, USA). Membranes were then incubated with peroxidase-linked anti-mouse or anti-rabbit secondary antibodies (1:5,000; Cell Signaling Technology, USA) for 2 h at room temperature. The expression levels of indicated proteins were quantified using quantity one.
8
Antibody Utilization in Signaling Pathway Analysis
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The following antibodies were purchased from Cell Signaling Technology: anti-phospho-tyrosine (p-Tyr-100), anti-Pim1, anti-Pim2, anti-Pim3, anti-Cyclin D3, anti-phospho-eIF4B, anti-AKT, anti-phospho-AKT (S473), anti-phospho-AKT (T308), anti-p70S6K, anti-phospho-p70S6K (T389), anti-phospho-S6 (S240/244), anti-phospho-GSK3β (S9), and anti-c-Myc, and anti-phospho IRS1 (S1101, S318, S302, S307, S636/639, S612) antibodies which recognize the following residues in human (h) or mouse (m) IRS1; anti-phospho IRS1 hS1101 (m1097); mS318 (hS323); mS302 (hS307); mS307 (hS312); hS636/S639 (mS632/635); mS612 (hS616). This numbering refers to human IRS1 (P35568); and mouse IRS1 (P35569). Anti-IRS1 (R & D systems), anti-RPS6 (Santa Cruz Biotechnology), anti-His (GenScript), anti-HA and anti-β-actin (Sigma) antibodies were used in these studies. Horseradish peroxidase (HRP)-linked enhanced chemiluminescence (ECL), mouse IgG and rabbit IgG secondary antibodies were purchased from GE Healthcare Life Sciences. Various protein kinase inhibitors, GNE-652 (Genentech), LGB321 (Novartis), AZD1208, AZD5363 (AstraZeneca) and PP242 (Selleck Chemicals) were used in these studies. All other chemicals including cycloheximide (CHX) and PMA were purchased from Sigma. Human recombinant insulin and insulin-like growth factor 1 (IGF1) were obtained from Peprotech.
9
Western Blot Analysis of Protein Expression
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HK1, HNE1, and CNE2 cell lines were transfected as described above. After 48 h transfection, proteins were extracted in the defined volume of RIPA lysis buffer containing 1.0 mM PMSF, protease inhibitor cocktail and DTT, and 50 μg proteins was loaded in SDS-PAGE gel electrophoresis10 (link). The proteins were transferred to PVDF Membrane (Milipore, Darmstadt, Germany) which were incubated with following primary antibodies: anti-HBP1 (Milipore, Darmstadt, Germany), anti-ZO-1, anti-E-cadherin, anti-N-cadherin, anti-Vimentin, anti-β-catenin, anit-MMP9, anti-NF-κB, anti-caspase 3 and cleaved caspase 3, anti-caspase 7, anti-PARP and cleaved PARP, anti-caspase 9 and cleaved caspase 9, anti-p18INK4C (p18), anti-p21Waft/Cip1 (p21), anti-p27Kip1 (p27), anti-Cyclin D1, anti-Cyclin D3, anti-CDK2, anti-CDK4, anti-CDK6 (Cell Signaling Technology, Danvers MA, USA), and β-actin (ABclonal, Cambridge, MA, USA) as an internal reference. Then the PVDF Membranes were incubated with HRP-linked anti-Rabbit or anti-Mouse IgG antibody according to the isotypes of the primary antibody. The membrane was imaged using ChemiDoc MP System (Bio-Rad, Hercules, CA, USA) and the images were analyzed using Image Lab Software (Bio-Rad, CA, USA).
10
Protein Expression Analysis in Cancer Cells
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Protein was extracted from HCT15 and A549 cells using CytoBuster Protein Extraction Reagent (Novagen, Madison, WI, USA) following the manufacturer's instructions. Equal amounts of protein (30 μg) were separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels and transferred to a 0.2 μm nitrocellulose membrane (Whatman GmbH, Hahnestraße, Germany). The membranes were blocked by incubating with Tris-buffered saline-Tween 20 (TBST) containing 10% skim milk (Difco, Sparks, MD, USA) for 2 h at room temperature. The blocked membranes were probed with anti-cyclin D3 (1:2000, 2936; Cell Signaling Technology, Danvers, MA, USA), anti-CDK2 (1:1000, sc-163; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p21 (1:1000, sc-397; Santa Cruz Biotechnology) or anti-p27 (1:500, sc-528; Santa Cruz Biotechnology) antibody at 4°C overnight. Membranes were incubated with horseradish peroxidase-conjugated horse anti-mouse IgG antibody (Vector Laboratories Inc., Burlingame, CA, USA) or goat anti-rabbit IgG antibody (Millipore, Billerica, MA, USA) for 1.5 h at room temperature. β-actin (Cell Signaling Technology) was used as an internal control.
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