P rb s807 811

Manufactured by Cell Signaling Technology

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The P-Rb S807/811 is a product offered by Cell Signaling Technology that detects phosphorylation of the retinoblastoma protein (Rb) at serine residues 807 and 811. This phosphorylation event is an important regulatory mechanism that modulates the activity of the Rb protein, which plays a crucial role in cell cycle control and proliferation.

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S807/811 (4 citations)

12 protocols using p rb s807 811

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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Immunoblotting Protocol for Protein Analysis

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Cells were lysed in lysis buffer (20 mM Tris-HCl [pH 7.4], 5 mM EDTA [pH 8.0], 10 mM Na₄P₂O₇, 100 mM NaF, 2 mM Na₃VO₄, 1% NP-40) containing aprotinin, leupeptin, PMSF, and phosphatase inhibitors cocktail 3 (Sigma). Protein samples were separated on 10% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated with primary antibody overnight at 4˚C. Primary antibodies against the following proteins were obtained from the indicated suppliers: C/EBPβ, Sestrin2, phospho-p70S6K (T389), p70S6K, p-Rb (S807/811) were from Cell Signaling Technology; Rb was from Santa Cruz Biotechnology; and β-actin (1:5,000) from Sigma.-Membranes were washed three times with TBST and incubated with HRP-conjugated anti-mouse (Santa Cruz Biotechnology) or anti-rabbit secondary antibody (Cell Signaling Technology). HRP was detected using the ECL reagent (BioNote).

Lee S., Byun J.K., Park M., Woo Kim S., Lee S., Kim J.G., Lee I.K., Choi Y.K, & Park K.G. (2020). Melatonin inhibits vascular smooth muscle cell proliferation and apoptosis through upregulation of Sestrin2. Experimental and Therapeutic Medicine, 19(6), 3454-3460.

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Western Blot Analysis of Cellular Signaling

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Cells were treated as indicated and western blot analysis was performed as previously described [2 (link)]. The pS6 S235/236 (4858), pS6 S240/244 (2215), S6 (2317), 4EBP1 (9644), pRb S780 (8180), pRb S807/811 (8516), Rb (9309), pBAD S112 (5284), p-p70S6K T389 (9205), p70S6K (2708), PIM1 (3247), PIM2 (4730), PIM3 (4165), TSC2 (4308), pGSK3β S9 (5558), GSK3β (9315), pPRAS40 T246 (2997), PRAS40 (2691), and GAPDH (2118) primary antibodies were purchased from Cell Signaling Technology, while the CDK4 (ab75511) and CDK6 (ab124821) antibodies were from Abcam and β-actin (A5441) was from Sigma-Aldrich. IRDye secondary antibodies were from LI-COR.

Litchfield L.M., Boehnke K., Brahmachary M., Mur C., Bi C., Stephens J.R., Sauder J.M., Gutiérrez S.M., McNulty A.M., Ye X.S., Wu W., Lallena M.J., Gong X., Merzoug F.F., Jansen V.M, & Buchanan S.G. (2020). Combined inhibition of PIM and CDK4/6 suppresses both mTOR signaling and Rb phosphorylation and potentiates PI3K inhibition in cancer cells. Oncotarget, 11(17), 1478-1492.

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Comprehensive DNA Damage Signaling Assay

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GFP (Abcam catalog no. ab6556), TRF1(Abcam catalog no. ab10579), GAPDH (Santa Cruz catalog no. sc-47724), OGG1 (Abcam catalog no. ab124741), Actin Cell Signaling catalog no. 3700), Lamin B1 Abcam catalog no. ab16048), Lamin A/C (Cell Signaling catalog no. 4777), γH2AX (Santa Cruz catalog no. sc-517348), 53BP1 (Novous catalog no. NB100-304), TRF2 (Novous catalog no. NB110-57130), MDM2 (Cell Signaling catalog no. 86934), p53(Santa Cruz catalog no. sc-126), p21 (Cell Signaling catalog no. 2947), p16 (Proteintech catalog no. 10883-1-AP), pRB S807/811 (Cell Signaling catalog no. 8516), pCHK2 T68(Cell Signaling catalog no. 2197), pCHK1 S317 (Cell Signaling catalog no. 12302), pATM S 1981 (Abcam catalog no. ab81292), CHK1 (Cell Signaling catalog no. 2360), H3K27me3 (Cell Signaling catalog no. 9733), H3K27Ac (Cell Signaling catalog no. 8173), LSD1 (Cell Signaling catalog no. 2184), cGAS (Cell Signaling catalog no. 66546), p62 (Cell Signaling catalog no. 39749).

Barnes R.P., de Rosa M., Thosar S.A., Detwiler A.C., Roginskaya V., Van Houten B., Bruchez M.P., Stewart-Ornstein J, & Opresko P.L. (2022). Telomeric 8-oxo-guanine drives rapid premature senescence in the absence of telomere shortening. Nature Structural & Molecular Biology, 29(7), 639-652.

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Western Blot Analysis of Cell Signaling

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Total protein from HCT116 cells were extracted using standard methods (Feng et al., 2018 (link)) and protein concentrations were determined by BCA protein assay kit (Thermo scientific, USA). A total of 40 µg of protein were separated by SDS-PAGE. Separated proteins were transferred onto a NC (nitrocellulose) membrane at 200 mA for 2 h. After transfer, membranes were blocked with 5% BSA in 1X PBST (phosphate-buffered saline with 0.1% Tween 20) at room temperature for 4 h. The membrane was probed with p53 (Zsbio, CN), Bcl-2 (Abcam, USA), Pro-caspase3 (Cell Signaling Technology, USA), Rb (Cell Signaling Technology, USA), pRb^s780 (Cell Signaling Technology, USA), pRb^s807/811 (Cell Signaling Technology, USA) or β-actin primary antibodies (Zsbio, CN) overnight at 4˚C and then secondary antibody at RT for 1 h according to manufacturer's instructions.

Li Y., Li Y., Li D., Li K., Quan Z., Wang Z, & Sun Z. (2020). Repositioning of Hypoglycemic Drug Linagliptin for Cancer Treatment. Frontiers in Pharmacology, 11, 187.

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Protein extraction and immunoblotting

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As previously described^{19 (link)}, cells were harvested in protein lysis buffer (1% Triton-X 100, 1% sodium deoxycholate, 0.1% SDS,150 mM NaCl, 10 mM EDTA, 20 mM Tris-Hcl pH 7.5, 2 M urea). After scraping the cells on ice with the lysis buffer, samples were briefly sonicated for 10 min at high speed to disrupt DNA-protein complexes. The total amount of protein present in each sample was measured using Pierce BCA Protein assay kit (Thermo Scientific Fisher). Prior to loading the samples on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), they were denatured in 6× Laemmli buffer at 95 °C for 5 min. Equal amounts of protein samples were separated by SDS-PAGE. This was followed by transfer on a nitrocellulose membrane and visualization with the following antibodies: pH2AX (S139) (9718, Cell Signalling), Beta-Actin (ab8227, Abcam), p21 (2947, Cell Signalling), pRb (S807/811) (9308, Cell Signalling), Rb (9309, Cell Signalling), MDM2 (OP 46, Calbiochem), p53 (DO-1, sc-126, Santa Cruz), p53-HRP (DO-1, sc-126, Santa Cruz), CDK4 (ab68266 abcam; DCS-35, sc-23896, Santa Cruz), p53 K382ac (2525, Cell Signalling) and Cyclin D1 (ab134175, Abcam).

Sriraman A., Dickmanns A., Najafova Z., Johnsen S.A, & Dobbelstein M. (2018). CDK4 inhibition diminishes p53 activation by MDM2 antagonists. Cell Death & Disease, 9(9), 918.

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Antibody and Chemical Analysis of Cell Cycle Regulation

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The following antibodies were used: pRB-S807/811 (Cell Signaling Technology, Danvers, MA), pRBL2-S672, RBL2 (Abgent, Inc., San Diego, CA), CCND1, CDK4, CDK6, GAPDH, β-ACTIN (Santa Cruz, Dallas, TX), RB (BD Biosciences, San Jose, CA), and γH2AX (Millipore, Billerica, MA). The following chemicals were used: PD-0332991 (Selleck Chemicals, Houston, TX), hydroxyurea, cytarabine, and aphidicolin (Sigma, St. Louis, MO).

Mohanty S., Mohanty A., Sandoval N., Tran T., Bedell V., Wu J., Scuto A., Murata-Collins J., Weisenburger D.D, & Ngo V.N. (2016). Cyclin D1 Depletion Induces DNA Damage in Mantle Cell Lymphoma Lines. Leukemia & lymphoma, 58(3), 676-688.

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Tissue Pulverization and RNA/Protein Analysis

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Frozen tissues were pulverized prior to protein extraction essentially as above. Total RNA was extracted using the Direct-zol RNA isolation kit (Genessee Scientific) per manufacturer's instructions. mRNA expression was detected as above. IHC analysis was conducted as described (13 (link)). Using antibodies purchased from Thermo Scientific (ESR1, RM-9101-S), (Ki67, RM-9106-S1) and Cell Signaling (pRb S807/811, 8516).

Wardell S.E., Ellis M.J., Alley H.M., Eisele K., VanArsdale T., Dann S.G., Arndt K.T., Primeau T., Griffin E., Shao J., Crowder R., Lai J.P., Norris J.D., McDonnell D.P, & Li S. (2015). Efficacy of SERD/SERM Hybrid-CDK4/6 inhibitor combinations in models of endocrine therapy resistant breast cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(22), 5121-5130.

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Immunohistochemical Profiling of Xenograft Tumors

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Fixed xenograft tumors were embedded in paraffin (FFPE). Unstained sections were stained with the following Abs purchased from Cell Signaling Technology: p-ERK1/2 T202/Y204 (catalog 4370) and p-Rb S807/811 (catalog 8516). The staining kit for Ki67 (catalog VP-K451; Vector Laboratories) was used per manufacturer’s instructions. Representative images were taken using a DM1000 LED light microscope camera (Leica Camera).

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Comprehensive Molecular Profiling Protocol

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Trametinib (GSK1120212) (#S2673), PLX4720 (#S1152), PLX4032 (#S1267), PLX8394 (#S7965), AZD5991 (#S8643), MK2206 (#S1078), buparlisib (BKM120) (#S2247), and Navitoclax (#S1001) were purchased from Selleck Chemicals, LLC. DRP-1 (#8570S), BAK (#12105S), Bcl-XL (2762S), Caspase 9 (#9502S), Cleaved caspase 3 (#9661S), HSP90 (#4877S), pERK1/2 (#9101S), pRb S807/811 (#9308S), pT308 AKT (#2965S), pPRAS40 T246 (#2640S), VCAM1 (#13662S), Mcl-1 (#4572S), LAMP-1 (#9091T), Vinculin (#4650S), Tubulin (#2148S), COX-IV (#11967S), cytochrome c (#11940S) cleaved PARP (#9541) and GAPDH (#2118S) antibodies were purchased from Cell Signaling Technology. ERK2 (sc-1647) and AIF (#sc-13116) were purchased from Santa Cruz Biotechnology Inc., γH2AX (ab11174) was purchased from Abcam, Actin (A2066) was purchased from Sigma-Aldrich Co., and Bcl-2 (#51-6511GR) was purchased from BD Biosciences. Goat anti-rabbit IgG Alexa Fluor^TM 488 secondary antibody was purchased from Invitrogen.

Cavallo M.R., Yo J.C., Gallant K.C., Cunanan C.J., Amirfallah A., Daniali M., Sanders A.B., Aplin A.E., Pribitkin E.A, & Hartsough E.J. (2024). Mcl-1 mediates intrinsic resistance to RAF inhibitors in mutant BRAF papillary thyroid carcinoma. Cell Death Discovery, 10, 175.

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