Antibodies used for flow cytometry were as follows: (anti-mouse) CD11b (M1/70), Gr1 (RB6-8C5), CD71 (R17217), Ter119 (TER-119), NK1.1 (PK136), CD45R (RA3-6B2), CD3 (17A2), CD19 (6D5), CD4 (GK1.5), cKit (2B8), Sca1 (D7), CD150 (mShad150), CD48 (HM48-1), CD16/32 (93), CD45.1 (A20), and CD45.2 (104). All anti-mouse antibodies were purchased from BioLegend, eBioscience or BD Biosciences. The “lineage cocktail” included NK1.1, CD11b, CD45R, CD3, Gr1, Ter119, CD19, and CD4. (anti-human) CD14 (61D3, eBioscience) and CD15 (HI98, BioLegend). Antibodies used for phospho-flow analysis were as follows: (primary antibodies) Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb (Cell Signaling), Phospho-Akt (Ser473) (D9E) XP Rabbit mAb (Cell Signaling), Phospho-S6 Ribosomal Protein (Ser235/236) Antibody (Cell Signaling). (secondary antibody) Allophycocyanin (APC) AffiniPure F(ab’)2 Fragment Donkey Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories)
Phospho s6 ribosomal protein ser235 236 antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The Phospho-S6 Ribosomal Protein (Ser235/236) Antibody is a tool used to detect the phosphorylation of the S6 ribosomal protein at serine residues 235 and 236. It can be used in various applications such as Western blotting, immunohistochemistry, and flow cytometry to study the activation of the mTOR signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Affinipure f ab 2 fragment donkey anti rabbit igg h l, by Jackson ImmunoResearch (1 mentions)
Phospho p44 42 mapk, by Cell Signaling Technology (1 mentions)
Cd14 61d3, by Thermo Fisher Scientific (1 mentions)
Cd15 hi98, by BioLegend (1 mentions)
Phospho akt ser473 d9e xp rabbit mab, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Foxp3 transcription factor staining buffer kit, by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
Lsrii flow cytometer, by BD (1 mentions)
3 protocols using phospho s6 ribosomal protein ser235 236 antibody
1
Flow Cytometry Antibody Panel for Immune Cell Analysis
2
Western Blot Analysis of AKT/MAPK Signaling
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The cells were seeded in 48-well plates (~10 × 104 cells/well) and serum-starved overnight before treatment. The cells were washed with ice-cold PBS after corresponding treatments and then lysed with Laemmli buffer containing 50 mM DTT (Sigma-Aldrich, Bayswater, VIC, Australia). Total protein samples (~20 μg/sample) were fractionated by SDS-PAGE gel electrophoresis and transferred onto Polyvinylidene Fluoride (PVDF) membrane (Merk Millipore Ltd., County Cork, Ireland). The membrane was probed with different primary antibodies, including mouse AKT/MAPK Signaling Pathway Antibody Cocktail (ab151279; Abcam), Phospho-S6 Ribosomal Protein (Ser235/236) Antibody (2211; Cell Signaling Technology), and mouse anti-GAPDH antibody (sc-47724; Santa Cruz). The membranes were then incubated with the appropriate IRDye680 or IRD800 labelled secondary antibody, imaged, and analysed using the ODYSSEY Infrared Imaging System (LI-COR, Lincoln, NE, USA).
3
Quantifying T Regulatory Cells and pS6 Expression
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A total of 5 × 105–1 × 106 cells were stained with antibodies listed in Supporting information, Table S1 . Tregs were detected by surface staining of CD4, CD25 followed by fixation, permeabilization and intracellular forkhead box protein 3 (FoxP3) staining using the FoxP3 transcription factor staining buffer kit (Thermo Fisher Scientific). For intracellular pS6 staining, cells were fixed with 4% paraformaldehyde and lysed with 0·1% saponin (Sigma‐Aldrich). pS6 was determined using phospho‐S6 ribosomal protein (Ser235/236) antibody (Cell Signaling Technology, Danvers, MA, USA) and detected by using a fluorescein isothiocyanate (FITC)‐coupled anti‐rabbit secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA). Flow cytometric analyses were performed on a LSR II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
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