Anti ps6

Manufactured by Cell Signaling Technology

Sourced in United States

Anti-pS6 is a primary antibody product offered by Cell Signaling Technology. It is designed to detect phosphorylated ribosomal protein S6, a key component of the cell's protein synthesis machinery.

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Lab products found in correlation

Close spelling variants of this product

Rabbit anti-pS6 (20 citations) Anti-pS6 Ser235/236 (14 citations) Anti-pS6 antibody (12 citations) Anti-pS6(Ser240/244) (7 citations) Anti-pS6 Ribosomal Protein (6 citations) Rabbit anti-pS6 Ser235/236 (5 citations) Rabbit anti-pS6-Ser240/244 (5 citations) Mouse anti-pS6 (5 citations) Rabbit anti-pS6 (S235/236, (4 citations) Anti-pS6 (clone D57.2.2E) (3 citations)

50 protocols using anti ps6

Immunostaining of Kidney Sections and Cultured Podocytes

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Kidneys were frozen in Optimum Cutting Temperature compound (Sakura) and sectioned at 3 μm thickness (Leica Kryostat). The sections were fixed in 4% paraformaldehyde for 15 minutes, permeabilized in 0.5% Triton X-100 in PBS for 5 minutes at room temperature, and then blocked in 2% normal donkey serum for 60 minutes. The sections were then immunostained with the following antibodies: anti-WT1 (catalog BS91456, Bioworld), anti-nephrin (Progen), anti-PP2Acα (catalog ab106262, Abcam), and anti–p-s6 (catalog 4858, Cell Signaling Technology).
The cultured podocytes seeded on coverslips were fixed in cold methanol/acetone (1:1) for 10 minutes at –20°C. After 3 extensive washes with 1× PBS, cells were treated with 1% Triton X-100 for 5 minutes, blocked in 2% normal donkey serum in 1× PBS buffer for 40 minutes at room temperature, and incubated with the following antibodies: anti–p-s6 (catalog 4858, Cell Signaling Technology), anti-CFB (catalog ab192577, Abcam), and anti–p-STAT1 (Ser727) (catalog 8826S, Cell Signaling Technology), followed by staining with FITC- or tetramethylrhodamine-conjugated secondary antibodies: Cy3-AffiniPure Donkey Anti-Rabbit IgG (H+L): 711-165-152, Jackson ImmunoResearch. Cells were also stained with DAPI to visualize the nuclei. Slides were viewed using an OLYMPUS DP74 and BX53 epifluorescence microscope equipped with a digital camera.

Lu Q., Hou Q., Cao K., Sun X., Liang Y., Gu M., Xue X., Zhao A.Z, & Dai C. (2021). Complement factor B in high glucose–induced podocyte injury and diabetic kidney disease. JCI Insight, 6(19), e147716.

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Protein Expression Analysis in Tumor Tissues

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Cells were lysed using boiling hot SDS lysis buffer (1.1% SDS, 11% glycerol, 0.1 mol/L Tris, pH 6.8) with 10% β-mercaptoethanol. Tumor tissue was crushed using a tissue lyser (TissueLyser II, QIAGEN) and cells were gently lysed using Triton X-100. Blots were probed with anti-α-tubulin (Merck), anti-HTR4 (ThermoFischer), anti-cleaved Caspase 8, anti-Akt, anti-p-Akt (Ser 473), anti-S6, anti-p-S6 (Ser235/6, Ser240/4), anti-p70 S6, anti-p-p70 S6 (Thr421/Ser424), anti-p-ERK1/2, anti-ERK1/2, anti-p-CREB (Ser133) and anti-CREB (all from Cell Signaling) and detected using the Odyssey infrared imaging system (Odyssey Fc, LI-COR Biosciences). Immunoblots were quantified using ImageJ.

Liu W., Stachura P., Xu H.C., Umesh Ganesh N., Cox F., Wang R., Lang K.S., Gopalakrishnan J., Häussinger D., Homey B., Lang P.A, & Pandyra A.A. (2020). Repurposing the serotonin agonist Tegaserod as an anticancer agent in melanoma: molecular mechanisms and clinical implications. Journal of Experimental & Clinical Cancer Research : CR, 39, 38.

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Analyzing Protein Expression in Glioblastoma Cells

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Western blotting was used to analyze protein expression according to standard procedures [24 (link)]. After various treatments, glioblastoma cells were lysed with iced RIPA buffer [25 (link)]. The isolated proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) thereby transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Blocked with 5% non-fat dried milk, the membranes were subsequently incubated with appropriate primary and secondary antibodies. The primary antibodies used in this study included anti-caspase-3 (Proteintech, Wuhan, China), anti-S6 (Cell Signaling Technology), anti-p-S6 (Cell Signaling Technology), anti-Akt (Cell Signaling Technology), anti-p-Akt (Ser 473, Cell Signaling Technology), anti-p-S6K1 (T389, Abcam), and anti-Actin (Cell Signaling Technology).

Wu Y., Li Z., Zhang L, & Liu G. (2019). Tivantinib Hampers the Proliferation of Glioblastoma Cells via PI3K/Akt/Mammalian Target of Rapamycin (mTOR) Signaling. Medical Science Monitor : International Medical Journal of Experimental and Clinical Research, 25, 7383-7390.

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Immunohistochemistry and Apoptosis Analysis

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Sections (5 um thick) were processed and endogenous peroxidase blocked. Incubations with anti-pS6 (1:200; cell signaling), anti-UBE2C (1:200; Boston Biochem) and anti-Ki-67 (1:50; Dako) were at 4°C overnight. Antigen was detected with 3,3-diaminobenzidine and counterstaining with hematoxylin. For TUNEL staining, ApopTag® Fluorescein In Situ Apoptosis Detection Kit (MILLIPORE) was used.

Kato M., Banuelos C.A., Imamura Y., Leung J.K., Caley D.P., Wang J., Mawji N.R, & Sadar M.D. (2015). Co-targeting Androgen Receptor Splice Variants and mTOR signaling pathway for the Treatment of Castration-Resistant Prostate Cancer. Clinical cancer research : an official journal of the American Association for Cancer Research, 22(11), 2744-2754.

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Immunofluorescence Labeling of Neural Markers

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First, free-floating sections were washed in PBS three times, followed by incubation in blocking buffer (0.1% Triton X-100, 1% bovine serum albumin, 5% normal goat serum in PBS) for 1 h. Then sections were incubated with primary antibody overnight at 4°C, washed for three times in PBS, and then incubated with secondary antibody for 2 h at 4°C. After 4′,6-diamidino-2-phenylindole (DAPI) treatment for 2 min, sections were washed again with PBS three times and mounted with Fluoromount-G (SouthernBiotech, Birmingham, AL). For BrdU incorporation, sections were treated before standard blocking protocol as follows: incubation with 2N HCl was performed at room temperature for 1 h, followed by soak in 0.1 m (pH 8.4) borate buffer for 10 min. After three washes in PBS, sections were processed according to standard protocol in blocking solution. The primary antibodies and their final concentrations used in the experiments were as follows: anti-BrdU (1:200, rat; AbD Serotec, Raleigh, NC), anti-pS6 (1:200, rabbit; Cell Signaling Technology, Danvers, MA), anti-GFP (1:1000, chicken; Abcam, Cambridge, MA), and anti-Sox2 (1:1000, goat; R&D Systems, Minneapolis, MN). Secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA) and applied in a 1:1000 dilution.

Wang X., Seekaew P., Gao X, & Chen J. (2016). Traumatic Brain Injury Stimulates Neural Stem Cell Proliferation via Mammalian Target of Rapamycin Signaling Pathway Activation. eNeuro, 3(5), ENEURO.0162-16.2016.

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Quantitative Western Blot Analysis of Telomeric Proteins

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Nuclear Cytosolic Fractionation Kit (BioVision) was used to obtain protein extracts. Protein concentration was determined using the Bio‐Rad DC Protein Assay (Bio‐Rad). Up to twenty micrograms of nuclear protein extracts was separated in SDS–polyacrylamide gels by electrophoresis. After protein transfer onto nitrocellulose membrane, the membranes were incubated with the indicated antibodies. Antibody binding was detected after incubation with a secondary antibody coupled to horseradish peroxidase using chemiluminescence with ECL detection KIT (GE Healthcare).
Primary antibodies: anti‐TRF1 1:1,000 (BED5, Bio‐Rad), anti‐TRF1 1:500 (homemade), anti‐SMC‐1 1:2,000 (Bethyl), anti‐AKT1 1:500 (Millipore), anti‐p‐AKT 1:500 (Ser473, Cell Signaling Cell Signaling Technology), anti‐S6 1:500 (Cell Signaling Cell Signaling Technology), anti‐p‐S6 1:500 (Ser240/244, Cell Signaling Technology), anti‐TIN2 1:1,000 (Abcam), anti‐RAP1 1:1,000 (Bethyl).
For the quantification, protein‐band intensities have been quantified by densitometric analysis with ImageJ software. The Trf1 total levels have been normalized versus SMC1 and the mean of the Trf1/SMC1 ratio deriving from at least 3 different replicates has been used to generate the chart.

Bejarano L., Bosso G., Louzame J., Serrano R., Gómez‐Casero E., Martínez‐Torrecuadrada J., Martínez S., Blanco‐Aparicio C., Pastor J, & Blasco M.A. (2019). Multiple cancer pathways regulate telomere protection. EMBO Molecular Medicine, 11(7), e10292.

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Immune Signaling Profiling in COVID-19 Recovery

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PBMCs from COVID-19 recovered patients and healthy donors were incubated with 10 μg/ml biotin-F(ab′)₂ anti-human Ig(M + G) on ice for 30 min, plus 20 μg/ml streptavidin on ice for 10 min, and then activated at 37 °C for indicated times. Cell lysates were used for electrophoresis in SDS–polyacrylamide gel, electrotransferred onto a nitrocellulose membrane, and then probed with the following specific antibodies: anti-pCD19 (Cat# 3571S, Cell Signaling Technology, USA), anti-CD19 (Cat# 90176, Cell Signaling Technology, USA), anti-pBtk (Cat# ab52192, Abcam, USA), anti-Btk (Cat# 8547S, Cell Signaling Technology, USA), anti-pPI3K (Cat# 4228S, Cell Signaling Technology, USA), anti-pAkt (Cat# 4060L, Cell Signaling Technology, USA), anti-pFoxO1 (Cat# 9461S, Cell Signaling Technology, USA), anti-pmTOR (Cat# 5536S, Cell Signaling Technology, USA), and anti-pS6 (Cat# 4856S, Cell Signaling Technology, USA). After incubated relative secondary antibodies, immunoreactive bands were presented and captured with the ChemiDoc XRS + imaging systems (Bio-Rad). β-actin or GAPDH was used as the loading control.

Jing Y., Luo L., Chen Y., Westerberg L.S., Zhou P., Xu Z., Herrada A.A., Park C.S., Kubo M., Mei H., Hu Y., Lee P.P., Zheng B., Sui Z., Xiao W., Gong Q., Lu Z, & Liu C. (2021). SARS-CoV-2 infection causes immunodeficiency in recovered patients by downregulating CD19 expression in B cells via enhancing B-cell metabolism. Signal Transduction and Targeted Therapy, 6, 345.

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Western Blot Analysis of Protein Expression

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Cell lysates were prepared by suspending cells in lysis buffer (PBS, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride). The lysates were cleared by centrifugation and subjected to electrophoresis on a sodium dodecyl sulfate–polyacrylamide gel. The proteins were then transferred to nitrocellulose membranes, blocked with 1–10% dry milk (or bovine serum albumin) in TBS-T buffer [TBS (10 mM Tris–HCl, pH 7.5, 135 mM NaCl) + 0.05% Tween-20]. Antigen–antibody complexes were visualized by chemiluminescence with an enhanced chemiluminescence substrate. Anti-S6 (1:1000), anti-pS6 (1:1000), anti-actin (1:1000) antibodies were purchased from Cell Signaling Technology. Immunoblot images were cut and rearranged to remove irrelevant information; however, all lanes were obtained from the same blots with the same levels of exposure and contrast.

Kunisada Y., Eikawa S., Tomonobu N., Domae S., Uehara T., Hori S., Furusawa Y., Hase K., Sasaki A, & Udono H. (2017). Attenuation of CD4+ CD25+ Regulatory T Cells in the Tumor Microenvironment by Metformin, a Type 2 Diabetes Drug. EBioMedicine, 25, 154-164.

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Immunohistochemical Analysis of Kidney and Liver

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Sliced paraffin sections were immersed in xylene for deparaffinization, rehydrated to PBS^- from ethanol, and then heated in a microwave oven to 100°C for 15 min in 10 mM sodium citrate buffer (pH 6.0) to unmask antigens. The sections were incubated in 0.3% H₂O₂/methanol to destroy endogenous peroxidase activity, and blocked with 1% bovine serum albumin and 0.05% NaN₃ in PBS^- for 30 min at room temperature, and then incubated with a primary antibody overnight at 4°C [23 (link), 24 (link)]. The primary antibodies used for immunohistochemistry were anti-pERK1/2 (M8159, dilution 1:200; Sigma), anti-pS6 (#4858, dilution 1:100; Cell Signaling), anti-Ki67 (ab16667, dilution 1:100; Abcam). The sections were rinsed and incubated with a biotinylated anti-mouse and anti-rabbit IgG/IgA/IgM secondary antibody (Histofine; Nichirei Biosciences, Tokyo, Japan) for immunohistochemical staining. Immune reaction products were developed with 3,3′-diaminobenzidine. Cells with positive signals were counted in 10 random fields of either kidney or liver sections obtained from 6 rats in each group by a naive observer using a 40× objective. In the kidney, we counted positive cells in both normal and cystic structural areas.

Kugita M., Nishii K., Yamaguchi T., Suzuki A., Yuzawa Y., Horie S., Higashihara E, & Nagao S. (2017). Beneficial effect of combined treatment with octreotide and pasireotide in PCK rats, an orthologous model of human autosomal recessive polycystic kidney disease. PLoS ONE, 12(5), e0177934.

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Investigating Heavy Metal Toxicity in Cells

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The plumbous acetate [Pb(Ac)₂] for Pb²⁺ supply was purchased from Sigma–Aldrich. Dulbecco's Modified Essential Medium (DMEM) and FBS were purchased from GIBCO Invitrogen. The Lyso-Tracker Red probes for acidic lysosome staining were from Beyotime. Anti-GRP78 (glucose-regulated protein 78), anti-GRP94, phosphorylates eukaryotic initiation factor 2 (anti-peIF2a), anti-cleaved caspase-3, anti-light chain 3 (anti-LC3) and anti-pS6 were purchased from Cell Signaling Technology. Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase), BCL2-associated X protein (anti-Bax), B-cell lymphoma 2 (anti-BcL2) and anti-p70S6K (S6 kinase 1) antibodies were from Millipore. The p62 antibody was from Santa Cruz. Other reagents were of the highest purity available.

Sui L., Zhang R.H., Zhang P., Yun K.L., Zhang H.C., Liu L, & Hu M.X. (2015). Lead toxicity induces autophagy to protect against cell death through mTORC1 pathway in cardiofibroblasts. Bioscience Reports, 35(2), e00186.

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