Antibodies for immunoblotting were obtained from the following sources: rabbit anti‐GAPDH (Cell signaling 5174), rabbit anti‐SIRT1 (Cell Signaling 2028), rabbit anti‐phospho‐S6 Ser235/236 (Cell Signaling 4858), and rabbit anti‐S6 (Cell Signaling 2217). Isolated intestine, crypts, or ISCs were lysed in RIPA buffer supplemented with protease Inhibitor and phosphatase inhibitor (Santa Cruz). Proteins extracts were denatured by the addition of SDS loading buffer, boiled and resolved by SDS‐PAGE, and analyzed by immunoblotting with primary antibodies listed above. The band density of all blots was quantified by ImageJ software.
Rabbit anti s6
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-S6 is a polyclonal antibody that binds to the ribosomal protein S6, a key component of the 40S subunit of the eukaryotic ribosome. The antibody is produced by immunizing rabbits with a synthetic peptide corresponding to a region of the S6 protein.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti phospho s6 ser235 236, by Cell Signaling Technology (3 mentions)
Rabbit anti 4e bp1, by Cell Signaling Technology (3 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (2 mentions)
Rabbit anti akt, by Cell Signaling Technology (2 mentions)
Rabbit anti sirt1, by Cell Signaling Technology (1 mentions)
Rapamycin, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit fluoresceinisothiocyanate conjugated igg, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse texas red conjugated igg, by Santa Cruz Biotechnology (1 mentions)
Goat anti insulin a c 12, by Santa Cruz Biotechnology (1 mentions)
Chicken anti rabbit fluorescein isothiocyanate conjugated igg, by Santa Cruz Biotechnology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Aprotinin, by Cytiva (1 mentions)
Rabbit anti β catenin, by Cell Signaling Technology (1 mentions)
Anti hif 1α, by BD (1 mentions)
Rabbit anti ps6, by Cell Signaling Technology (1 mentions)
Rabbit anti p akt thr308, by Cell Signaling Technology (1 mentions)
M per, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Mouse anti actin, by MP Biomedicals (1 mentions)
Lumi light, by Roche (1 mentions)
10 protocols using rabbit anti s6
1
Immunoblotting with Intestinal Proteins
2
Immunochemical Analysis of Cellular Signaling Proteins
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Rabbit anti-S6, rabbit anti-phospho-S6 (ser235, 236), rabbit anti-4EBP-1, rabbit anti-phospho-4EBP-1 (Thr37, 46), rabbit anti-β-Catenin (species cross-reactivity: human, mouse, and rat) and mouse anti-proliferating cell nuclear antigen (PCNA) were from Cell Signaling Technology (Beverly, MA). Rabbit anti-α-Amylase was from Sigma Chemical Co. (St. Louis, MO). Mouse anti-CA19-9 was from Origene (Beijing, China). Goat anti-insulin A (C-12) (species cross-reactivity: human, mouse, and rat), Goat anti-glucagon (N-17) (species cross-reactivity: human, mouse, and rat), rabbit anti-somatostatin (FL-116) (species cross-reactivity: human, mouse, and rat), goat anti-rabbit fluoresceinisothiocyanate-conjugated IgG, donkey anti-goat fluoresceinisothiocyanate- conjugated IgG, donkey anti-goat Texas Red-conjugated IgG, goat anti-mouse Texas Red-conjugated IgG, and chicken anti-rabbit fluoresceinisothiocyanate-conjugated IgG and rapamycin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Dimethylsulfoxide was from Sigma Chemical Co. (St. Louis, MO). Aprotinin was purchased from Amersham Biosciences (Pittsburgh, PA). IRDye-conjugated affinity purified anti-rabbit, anti-mouse IgGs were purchased from Rockland (Gilbertsville, PA).
3
Western Blot Analysis of Breast Cancer Cells
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MCF-7 and MDA-MB-231 cells were lyzed in MPER (Thermo Scientific, Bleiswijk, The Netherlands) and diluted 1:1 with SDS sample buffer (4% SDS, 20% glycerol, 0.5 mol/l Tris-HCl (pH 6.8), 0.002% bromophenol blue). Lysates were resolved by SDS-PAGE and transferred to PVDF membranes. Membranes were incubated overnight at 4 °C and probed with the following antibodies: rabbit-anti-AKT, rabbit-anti-pAKT (Thr308), rabbit-anti-S6, rabbit-anti-pS6, rabbit-anti-4EBP1 (all Cell Signaling Technologies, Leiden, The Netherlands) in a 1:1000 dilution or anti-HIF1α (BD Biosciences, Breda, The Netherlands) and mouse-anti-actin (MP Biomedicals, Santa Ana, USA) in a 1:10,000 dilution. Primary antibodies were stained using HRP-coupled goat anti-rabbit or rabbit anti-mouse IgG and developed with Lumi-Light (Roche, Almere, The Netherlands). Images were captured with the ChemiDoc MP imaging system (Bio-Rad, Veenendaal, The Netherlands) and Image Lab Software.
4
Antibody Selection for Neurodegenerative Research
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Previously described procedures were followed for these experiments (43 (link), 44 (link)). The antibodies used were rabbit anti-AGAT (orb247515, Biorbyt); mouse anti-GAMT (sc-398936, Santa Cruz Biotechnology); mouse anti–synaptotagmin 1/2 (105011, Synaptic Systems); rabbit anti–PSD-95 (AB9708, Millipore); rabbit anti-Homer1 (ab211415, Abcam); rabbit anti–p-AMPK (Thr172) (2531S, Cell Signaling Technology); rabbit anti–p-UL K (Ser317) (12753, Cell Signaling Technology); rabbit anti-p62 rabbit (8025S, Cell Signaling Technology); rabbit anti–p-mTOR (Ser2448) (2971S, Cell Signaling Technology); rabbit anti-mTOR (2972S, Cell Signaling Technology); rabbit anti–p70 S6 kinase (Thr389) (9205S, Cell Signaling Technology); rabbit anti–p-S6 (Ser240/244) (5364S, Cell Signaling Technology); rabbit anti-S6 (2217S, Cell Signaling Technology); rabbit anti–p-4E-BP1(Thr37/46) (2855S, Cell Signaling Technology); rabbit anti–4E-BP1 (9644S, Cell Signaling Technology); rabbit anti–p-ULK (Ser757) (6888S, Cell Signaling Technology); rabbit anti-GAPDH (5174S, Cell Signaling Technology); rabbit anti-LC3B (ab48394, Abcam); and rabbit anti–cleaved caspase-3 (Asp175) (9661S, Cell Signaling Technology).
5
Western Blot Analysis of Protein Expression
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Protein extracts were prepared in RIPA buffer (10 mM Tris-HCl pH7.4, 150 mM NaCl, 1 mM EGTA, 0.5% Triton and complete 1% protease and phosphatase inhibitor mixture, Roche Diagnostics). Primary neurons, brain and liver lysate samples (50 μg protein lysates) were separated using 8% polyacrylamide gel and then transferred to PVDF membranes. Membranes were incubated overnight at 4°C with the following primary antibodies in 1X PBST with 5% w/v nonfat dry: rabbit anti-MeCP2 (1:1000; Sigma-Aldrich, RRID:AB_262075 ), mouse anti-V5 (1:1000; ThermoFisher Scientific, RRID:AB_2556564 ), rabbit anti-pS6 235/236 (1:500, Cell Signaling, RRID:AB_331679 ), rabbit anti-S6 (1:500, Cell Signaling, RRID:AB_945319 ), rabbit anti-Calnexin (1:50000, Sigma-Aldrich, RRID:AB_476845 ), mouse anti-β-Actin (1:50000; Sigma-Aldrich, RRID:AB_262137 ) or the mice serum (1:200) extracted through retro-orbital bleeding followed by centrifugation (10 min, RT, 13000 rpm). Subsequently, membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10000; Dako). The signal was then revealed with a chemiluminescence solution (ECL reagent, RPN2232; GE Healthcare) and detected with the ChemiDoc imaging system (Bio-Rad).
6
Comprehensive Immunoblotting Analysis
7
Immunofluorescence and Western Blotting Techniques
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The following primary antibodies were used for immunofluorescence and western blotting: mouse monoclonal anti-HA.11 ascites (1:500, Biolegend #901515), rabbit anti-pS6 S240/244 (1:500, Cell Signaling #2215), rabbit anti-NeuN (1:500, Cell Signaling #12943), rabbit anti-pS6 S235/236 (1:1000, Cell Signaling #2211), rabbit anti-S6 (1:1000, Cell Signaling #2217), rabbit anti-β-actin (1:1000, Cell Signaling #4970S). The following secondary antibodies were used for immunofluorescence and western blotting: goat anti-rabbit Alexa 488 (1:1000, Invitrogen #A11008) and goat anti-mouse Alexa 568 (1:1000, Invitrogen #A11031).
8
Western Blot Analysis of IL-17 Signaling
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Cells were lysed in RIPA lysis buffer (Cell Signaling Technology, Danvers, MA), normalized, subjected to SDS-PAGE, and blotted onto polyvinylidene fluoride membranes. After blocking in 5% milk and/or tris-buffered saline with Tween 20, membranes were probed with the indicated antibodies and visualized with horseradish peroxidaseeconjugated secondary antibodies using ECL Substrate (Bio-Rad, Hercules, CA). The following primary antibodies were used: mouse anti-actin antibody from Sigma; rabbit anti-IL17RB from Proteintech; rabbit anti-pS6 (S235/6), rabbit antiephosphorylated protein kinase B (S473), rabbit antiephosphorylated extracellular signaleregulated kinase 1/2 (T202/Y204), rabbit antiephosphorylated NF-kB p65, rabbit antieNF-kB p65, rabbit anti-S6, rabbit antieprotein kinase B, mouse antieextracellular signaleregulated kinase 1/2, mouse anti-STAT3, rabbit antiephosphorylated STAT3 (S727), and rabbit antiephosphorylated STAT3 (Y705) from Cell Signaling Technology; and rabbit anti-IL17RA from Abcam. ImageJ software was used to quantify the intensity of nonsaturated western blot bands. For mesoscale, RHE was lysed in RIPA buffer and IL-17E was measured using the U-PLEX Human IL-17E/IL-25 Assay set according to manufacturer's protocol (Meso Scale Diagnostics, Rockville, MD).
9
Ghrelin Peptide Signaling Pathway
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The ghrelin peptide was purchased from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, USA). Dexamethasone was purchased from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-β-actin and rabbit anti-S6 and anti-p-S6 were obtained from Cell Signaling Technology (Beverly, MA, USA). Rabbit anti-GHSR1a and rabbit anti-Clock were purchased from Santa Cruz Inc. (Santa Cruz, CA, USA). Rabbit anti-Per2 was from MBL Inc. (Medical & Biological Laboratories, Nagoya, Japan).
10
Antibody Panel for Cell Signaling Analysis
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The following primary antibodies (at the dilution indicated) were used for either immunofluorescence or Western blotting: mouse anti-p21 (1:100, cod. B1313; Santa Cruz, Biotechnology, Dallas, TX, USA); rabbit anti-Ki67 (1:100, cod. HPA001164; Sigma-Aldrich, St. Louis, MO, USA); rabbit anti-phospho-ERK1/2 (Thr202/Tyr204, Thr185/Tyr187) (1:500, cod. 05–797R; Millipore, Burlington, MA, USA); mouse anti-ERK1/2 (1:500, cod. 05–1152; Millipore, Burlington, MA, USA); rabbit anti-phospho-S6 (Ser235/236) (1:500, cod. 4856; Cell Signaling, Danvers, MA, USA); rabbit anti-S6 (1:500, cod. 2217; Cell Signaling, Danvers, MA, USA); mouse anti-N-Cadherin (1:50, cod. 610920; BD Biosciences, Franklin Lakes, NJ); mouse anti-E-Cadherin (1:50, cod. 610404; BD Biosciences, Franklin Lakes, NJ); rabbit anti-LC3 (1:1000, cod. L7543; Sigma-Aldrich, St. Louis, MO, USA); mouse anti-LAMP1 (1:1000, cod. 555798; BD, Biosciences, Franklin Lakes, NJ); rabbit anti- β-Catenin (1:500, cod. PA5-77934; Invitrogen, Paisley, UK); mouse anti-β-Actin (1:2000, cod. A5441; Sigma-Aldrich, St. Louis, MO, USA); mouse anti-β-Tubulin (1:1000, cod. T5326; Sigma-Aldrich, St. Louis, MO, USA); rabbit anti-GAPDH (1:1000, cod. G9545; Sigma-Aldrich, St. Louis, MO, USA).
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