Total rb

Manufactured by Cell Signaling Technology

Sourced in United States

Total Rb is a lab equipment product offered by Cell Signaling Technology. It is designed to detect and quantify the total levels of the Rb (Retinoblastoma) protein, which plays a critical role in cell cycle regulation.

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Close spelling variants of this product

Total/phosphorylated Rb (2 citations) Rb-PKM total (2 citations) Rb anti-total p70 S6K (2 citations)

15 protocols using total rb

Protein Expression and Cell Proliferation Analysis

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Western blot and RNA immunoprecipitation assay was performed as previously described[29 (link)]. Immunohistochemistry (IHC): Phospho-Rb, total Rb, Ki67 and TUNEL staining were performed on 10% paraffin embedded tumor slides per manufacture protocol guidelines and imaged using Leica florescence microscope at 20x and 40x magnifications. IHC staining was performed by our pathology department, Dr. Wei Jiang and Raymond O’Neill, at Thomas Jefferson University.
Western antibodies: Phospho-Rb (1:500, Cell Signaling Technology, Danvers, MA, catalog #9307), total Rb (1:500, Cell Signaling Technology, catalog #9309), HuR (1:1000, Life Technology/ Thermo Fisher, Waltham, MA, catalog# 390600), YAP1 (1A12) (1:500, Cell Signaling Technology, catalog# 12395), cyclin D1 (1:500, Santa Cruz, Santa Cruz, CA, catalog# 8396), p27 (1:1000, Santa Cruz, Santa Cruz, CA, catalog #sc-1641) and alpha-tubulin (1:5000, Invitrogen, Waltham, MA, catalog# A11126) primary antibodies used for protein evaluation by western blot.
Immunohistochemistry (IHC): Phospho-Rb (Cell signaling technology, catalog #8516), total Rb (Cell signaling Technology, catalog #9309) and Ki67 (Roche, Branchburg, NJ, catalog #790–4286) and TUNEL (Trevigen TACS 2 TdT Core Kit, Gaithersburg, MD, catalog# 4810-30-CK) staining antibodies were used for IHC analysis. For TUNEL staining, only diaminobenzidine (DAB) staining was used.

Dhir T., Schultz C.W., Jain A., Brown S.Z., Haber A., Goetz A., Xi C., Su G.H., Xu L., Posey J I.I.I., Jiang W., Yeo C.J., Golan T., Pishvaian M.J, & Brody J.R. (2019). Abemaciclib is effective against pancreatic cancer cells and synergizes with HuR and YAP1 inhibition. Molecular cancer research : MCR, 17(10), 2029-2041.

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Western Blot Antibody Panel

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AntiMDM2 antibody – 1:1:1 mix of mouse monoclonal antibodies 4B2, 2A9, 4B11; antip53 antibody - 1:1:1 mix of mouse monoclonal antibodies 240,421,1801; rabbit polyclonal antiActin (Sigma-Aldrich) or anti-Actin HRP (Santa Cruz); phospho Rb (Ser807/811: Cell Signaling); Total Rb (Cell Signaling); E2F1 (Cell Signaling) and secondary anti-mouse and anti-rabbit antibodies (Sigma-Aldrich) were used for western blot analysis.

Kundu N., Brekman A., Kim J.Y., Xiao G., Gao C, & Bargonetti J. (2017). Estrogen-activated MDM2 disrupts mammary tissue architecture through a p53-independent pathway. Oncotarget, 8(29), 47916-47930.

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Immunoblotting and Immunohistochemistry Protocols

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The following primary antibodies were used in this study for immunoblotting: pRB^ser780 (CST-3590), pRB^ser807 (CST-8516), total-RB (CST-9309), cyclin D1 (CST-2922), cyclin D3 (CST-2936), pAKT^ser473 (CST-9271), pAKT^Thr308 (CST-9275), total-AKT (CST-9272), pEGFR^Tyr1068 (CST-3777), total-EGFR (CST-2232), pERBB2^Tyr1248 (CST-2243), total-ERBB2 (CST-4290), pERBB3^Tyr1222 (CST-4784), pIGF1R^Tyr1135 (CST-3918), pS6K^Ser235/236 (CST-2211), total-S6K (CST-2217), Raptor (CST-2280), RheB (CST-13879), p4EBP1^Thr37/46 (CST-2855), 4EBP1 (CST-9452), pSIN1^Thr86 (CST-14716), SIN1 (CST-12860), pER^ser167 (CST-5587), Rictor (CST-2114) and Deptor (SCT-11816) were purchased from Cell Signaling Technology. p107 (sc-318), p130 (sc-317), total-ER (sc-8002, F-10), ERBB3 (sc-415) and IGF1R (sc-713) were purchased from Santa Cruz Biotechnology. β-tubulin (T-9026) were from Sigma-Aldrich and Ki67 from Clinisciences. The following antibodies were used for immunohistochemistry: pERK1/2^Thr202/4 (CST-4370), pAKT^ser473 (CST-4060), pS6K^Ser235/6 (CST-4858), pmTOR^Ser2448 (CST-2976) and p4EBP1^Thr37/46 (CST-2855) were purchased from Cell Signaling Technology. Ki67 was purchased from Clinisciences. Reagents were obtained from the following sources: 17-β-oestradiol (E2) and 4-hydroxytamoxifen (4-OHT) from Sigma-Aldrich, fulvestrant from Tocris, and neratinib and vistusertib from SelleckChem.

Pancholi S., Leal M.F., Ribas R., Simigdala N., Schuster E., Chateau-Joubert S., Zabaglo L., Hills M., Dodson A., Gao Q., Johnston S.R., Dowsett M., Cosulich S.C., Maragoni E, & Martin L.A. (2019). Combination of mTORC1/2 inhibitor vistusertib plus fulvestrant in vitro and in vivo targets oestrogen receptor-positive endocrine-resistant breast cancer. Breast Cancer Research : BCR, 21, 135.

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Comprehensive Immunoblot Analysis

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Immunoblot analysis was performed as previously described (7 (link)). Primary Abs against p-ERK1/2 T202/Y204 (catalog 4370), total ERK1/2 (catalog 4695), p-AKT S473 (catalog 4060), total AKT (catalog 9272), p-Rb S807/811 (catalog 8516), total Rb (catalog 9309), p-p90RSK T359/S363 (catalog 9344), RSK1 (catalog 8408), p-HER2/ErbB2 Y1221/1222 (catalog 2243), HER2/ErbB2 (catalog 2165), p-EGFR Y1068 (catalog 3777), cyclin D1 (catalog 2922), CDK4 (catalog 12790), CDK6 (catalog 13331), vinculin (catalog 18799), and β-actin (catalog 12620) were purchased from Cell Signaling Technology. Pierce HRP-conjugated secondary Abs (anti-rabbit and anti-mouse) were purchased from Thermo Fisher Scientific. Amersham ECL Prime chemiluminescent detection reagent (GE Healthcare Life Sciences) was used to visualize protein expression.

Li T., Kikuchi O., Zhou J., Wang Y., Pokharel B., Bastl K., Gokhale P., Knott A., Zhang Y., Doench J.G., Ho Z.V., Catenacci D.V, & Bass A.J. (2023). Developing SHP2-based combination therapy for KRAS-amplified cancer. JCI Insight, 8(3), e152714.

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Cell Cycle Protein Expression Analysis

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EPRS (Abcam ab31531); ESR1 (Cell Signaling 8644); phospho-RB (Cell Signaling 9301); total RB (Cell Signaling 9309); CDK2 (Cell Signaling 2546); CCNB1 (Cell Signaling 12231); GAPDH (Cell Signaling 2118), β-tubulin (Cell Signaling 2128), HSP70 (Cell Signaling 4872).

Katsyv I., Wang M., Song W.M., Zhou X., Zhao Y., Park S., Zhu J., Zhang B, & Irie H.Y. (2016). EPRS is a critical regulator of cell proliferation and estrogen signaling in ER+ breast cancer. Oncotarget, 7(43), 69592-69605.

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Cellular Assays Using Diverse Reagents

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Disposable cell culture ware was purchased from Corning Glass works (Corning, NY) and Zellkulture Flaschen (Europe, Switzerland). Media and other cell culture reagents were obtained from Mediatech, Inc (Herndon, VA) and Invitrogen (Long Island, NY). Dialyzed fetal bovine serum (dFBS), insulin, and Camptothecin were purchased from Sigma (St Louis, MO). Recombinant rat TNF- was obtained from BD PharMingen International (San Diego, CA). The Enhanced Chemiluminescence (ECL) Western Blot detection system was purchased from Perkin Elmer (Boston, MA). DFMO was a gift from ILEX Oncology^™ Inc, (San Antonio, TX). Phospho-p53 Ser15, total-p53, phospho-H2AX Ser139, phospho-Rb Ser 780, total-Rb, and cleaved active caspase-3 (Asp 175) antibodies were purchased from Cell Signaling (Beverly, MA). p21Cip1 antibody was purchased from BD Biosciences (San Diego, CA). AZD5438 (Cdk1, 2, and 9 inhibitor) and NU6140 (Cdk2 inhibitor) were purchased from Calbiochem, EMD Biosciences (La Jolla, CA). The Cell Death Detection ELISA Plus kit and WST-1 cell proliferation kit were purchased from Roche Diagnostics Corp. (Indianapolis, IN). The IEC-6 cell line (ATCC CRL 1592) and Caco2 (ATCC HTB-37) were obtained from American Type Culture Collection (Rockville, MD). All chemicals were of the highest purity commercially available.

Bhattacharya S., Ray R.M, & Johnson L.R. (2014). Cyclin-dependent kinases regulate apoptosis of intestinal epithelial cells. Apoptosis : an international journal on programmed cell death, 19(3), 451-466.

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Cell Cycle Regulator Protein Detection

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We purchased ribociclib, palbociclib, and abemaciclib from Selleck Chemicals (Houston, TX) and obtained alpelisib from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). In addition, fulvestrant was acquired from Sigma–Aldrich (St. Louis, MO), paclitaxel and U0126 were purchased from Cell Signaling Technology (Danvers, MA). Everolimus was obtained from LC laboratories, Inc. (Woburn, MA), and eribulin was acquired from Eisai (Tokyo, Japan).
In western blotting, the sources of antibodies were as follows: total Rb (#9309), pRb (Ser780; #3590), cyclin D1 (#2922), cyclin E1 (#20808), CDK2 (#2546), CDK4 (#12790), CDK6 (#13331), p21 (#2947), p27 (#2552) and β-tubulin (#2146)—all acquired from Cell Signaling Technology. In addition, we purchased horseradish peroxidase–conjugated secondary antibody from Bio-Rad Laboratories Inc. (Hercules, CA).

Iida M., Nakamura M., Tokuda E., Toyosawa D., Niwa T., Ohuchi N., Ishida T, & Hayashi S.I. (2019). The p21 levels have the potential to be a monitoring marker for ribociclib in breast cancer. Oncotarget, 10(47), 4907-4918.

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Western Blot Protein Analysis

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Proteins were extracted and blotted (30 μg) as described by Faião-Flores and collaborators (2016) (21 ). After analysis, Western Blots were stripped once and reprobed for β-actin or GAPDH to show even protein loading. Antibodies to phosphorylated RB S807/811, total RB, Parp, Caspase-9, Caspase-7, Bcl-2, H3K9me3 and CyclinB1 were purchased from Cell Signaling Technology (Beverly, MA, USA). The monoclonal antibodies to β-actin and GAPDH were purchased from Sigma (St. Louis, MO, USA). Monoclonal antibody to p21 was purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Massaro R.R., Faião-Flores F., Rebecca V.W., Sandri S., Alves-Fernandes D.K., Pennacchi P.C., Smalley K.S, & Maria-Engler S.S. (2017). Inhibition of proliferation and invasion in 2D and 3D models by 2-methoxyestradiol in human melanoma cells. Pharmacological research, 119, 242-250.

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Western Blot Analysis of Cell Cycle Proteins

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Cells were lysed in ice-cold whole cell lysis buffer(50 mM Tris–HCl, pH 8.0, 4 M urea and 1% Triton X-100) supplemented with complete protease inhibitor Cocktail (Roche Diagnostics, 04693132001). Whole cell lysates with equal protein were resolved by SDS-PAGE and transferred to nitrocellulose membrane. After blocking with 5% milk in Tris-buffered saline plus 0.02% Tween-20 (TBST), membranes were incubated with the following antibodies: cyclin D1 (Epitomics, 1677–1), cyclin D3 (Epitomics, 1846–1), E2F1 (Cell Signaling, 3742), cyclin E (Santa Cruz, sc-481), cyclin A (Santa Cruz, sc-596), CDK2 (Cell Signaling, 2546), CDK4 (Cell Signaling, 2906); phosphor-Rb (Ser 780) (Cell Signaling, 3590), phosphor-Rb (Ser-811) (ABclonal Biotechnology, AP0089), total Rb (Cell Signaling, 9309) and GAPDH (Proteintech, 10494-1-AP). Membranes were then incubated with horseradish peroxidase-coupled specific secondary anti-mouse (KPL, 074–1806) or anti-rabbit antibodies (KPL, 474–1506). Protein bands were visualized using ECL blotting detection reagents (KPL, 54-61-00).

Wu J., Lv Q., He J., Zhang H., Mei X., Cui K., Huang N., Xie W., Xu N, & Zhang Y. (2014). MicroRNA-188 suppresses G1/S transition by targeting multiple cyclin/CDK complexes. Cell Communication and Signaling : CCS, 12, 66.

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Western Blot Analysis of Signaling Proteins

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The western blot analysis was carried out as described by Park et al. [18 (link)]. The protein sample (20 μg) was loaded and separated by SDS–PAGE. After electrophoresis, the protein was electrotransferred to a poly vinylidene difluoride membrane (Immobilon-P; Millipore Corp., Billeria, MA). The antibodies phosphorylated Src (Y416, 1:500), total Src (1:1000), p-Rb (S780, 1:500 dilution), total Rb (1:1,000 dilution), p-IkBα (S32/36, 1:500 dilution), total IkBα (1:1,000 dilution), caspase 8 (1:1,000 dilution), and GAPDH (1:1,000 dilution) were purchased from Cell Signaling Technology (Denver, MA). Antibodies for VEGF (1:200 dilution) and TNFα (1:200 dilution) were purchased from Santa Cruz Biotechnology. Antibody for cyclin D1 (1:500 dilution) and TRβ (1:500) were purchased from Neomarkers (Thermo Scientific, Cambridge, MA) and Rockland, respectively. The blots were stripped with Re-Blot Plus (Millpore, Billeria, MA) and reprobed with rabbit polyclonal antibodies to GAPDH. Band intensities were quantified by using NIH IMAGE software (Image J 1.47).

Park J.W., Zhao L., Willingham M.C, & Cheng S.Y. (2016). Loss of Tyrosine Phosphorylation at Y406 Abrogates the Tumor Suppressor Functions of the Thyroid Hormone Receptor β. Molecular carcinogenesis, 56(2), 489-498.

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