Brain samples of mice were flash-frozen in liquid nitrogen and homogenized using an ultrasonic homogenizer in 2 × cell lysis buffer (Cell signaling, Danvers, MA, USA) supplemented with phosphatase inhibitor PhosSTOP (Sigma-Aldrich, St. Louis, MO, USA) and complete protease inhibitor (Sigma-Aldrich), according to the manufacturer’s instructions. Total protein samples (35 μg) were separated on 4–15% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and 4–12% Bolt™ Bis-Tris Plus gels (Thermo Fisher, Waltham, MA, USA) and transferred to polyvinylidene difluoride membranes (Bio-Rad/Thermo Fisher). The primary antibodies used were as follows: anti-Depdc5 (Abcam, Cambridge, UK, #ab185565, 1:1000), anti-βactin (8H10D10) (Cell signaling, #3700, 1:2000), anti-phospho-S6 Ribosomal Protein (Ser 240/244) (Cell signaling, #5364, 1:1000), anti-S6 Ribosomal Protein (Cell signaling, #14467, 1:1000), anti-phospho-Akt (ser473) (Cell signaling, #4060, 1:2000) and Akt (pan) (Cell signaling #2920, 1:2000). Signals were detected by chemiluminescence using ImageLab (Bio-Rad) and iBright CL 1500 (Thermo Fisher).
Anti phospho s6 ribosomal protein ser240 244
Manufactured by Cell Signaling Technology
Sourced in United Kingdom, United States
Anti-phospho-S6 ribosomal protein (Ser240/244) is a laboratory reagent used for the detection and analysis of phosphorylated S6 ribosomal protein. S6 ribosomal protein is a component of the 40S subunit of the eukaryotic ribosome and its phosphorylation is involved in the regulation of protein synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Anti s6 ribosomal protein, by Cell Signaling Technology (4 mentions)
Anti 4e bp1, by Cell Signaling Technology (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Complete protease inhibitor, by Merck Group (1 mentions)
Sds polyacrylamide gel, by Bio-Rad (1 mentions)
Anti β actin 8h10d10, by Cell Signaling Technology (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Pan akt, by Cell Signaling Technology (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Bolt bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
Ibright cl1500, by Thermo Fisher Scientific (1 mentions)
Phosstop, by Merck Group (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Anti phospho 4e bp1 thr37 46, by Cell Signaling Technology (1 mentions)
Anti phospho mtor ser2448, by Cell Signaling Technology (1 mentions)
Anti ubiquitin, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
6 protocols using anti phospho s6 ribosomal protein ser240 244
1
Quantification of Protein Signaling in Mouse Brain
2
Western Blotting of Zebrafish Proteins
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Western blotting was performed as described previously (Ding et al., 2016 (link)). Embryos from 6 days postfertilization (dpf) or fresh hearts isolated from adult fish were transferred immediately to RIPA buffer (Sigma-Aldrich) supplemented with complete protease inhibitor cocktail (Roche) and homogenized using a Bullet Blender tissue homogenizer (Next Advance). The resultant protein lysates were subjected to western blotting using a standard protocol. The following primary antibodies were used: anti-actin (1:8000, Santa Cruz Biotechnology, sc-1615), anti-phospho-mTOR (Ser2448) (1:2000, Cell Signaling Technology, 2971), anti-phospho-S6 ribosomal protein (Ser240/244) (1:5000, Cell Signaling Technology, 2215), anti-S6 ribosomal protein (1:8000, Cell Signaling Technology, 2217), anti-phospho-4E-BP1 (Thr37/46) (1:1000, Cell Signaling Technology, 2855), anti-4E-BP1 (1:2000, Cell Signaling Technology, 9644), anti-ubiquitin (1:1000, Thermo Fisher Scientific, PA5-17067) and anti-LC3 (1:3000, Novus Biologicals, NBP100-2331).
3
Protein Extraction and Western Blot Analysis
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Protein extracts were obtained using Nuclear Cytosolic Fractionation Kit (Biovision) or RIPA extraction buffer and protein concentration was determined using the Bio-Rad DC Protein Assay (Bio-Rad). Up to 20 μg of protein per extract were separated in SDS–polyacrylamide gels by electrophoresis. After protein transfer onto nitrocellulose membrane (Whatman), the membranes were incubated with the following primary antibodies: anti-S6 ribosomal protein (1:1000; Cell Signaling Technology, Cat#2217), anti-phospho-S6 ribosomal protein (Ser 240/244) (1:1000; Cell Signaling Technology, Cat#2215), anti LC3 (1:500; Cell Signaling Technology, Cat#2775), anti-β-actin (Sigma), anti-SMC1 (1:2000; Bethyl), anti-total OXPHOS (1:500; Total OXPHOS Rodent WB Antibody Cocktail, Cell Signaling Technology, Cat#110413). Antibody binding was detected after incubation with a secondary antibody coupled to horseradish peroxidase using chemiluminescence with ECL detection KIT (GE Healthcare). Protein-band intensities were measured with ImageJ software.
4
Antibody Panel for ErbB Receptor Signaling
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Anti-neu/HER2 (ErbB2) antibody was from Invitrogen. Anti-ErbB2, anti-ErbB3, anti-EGFR, anti-caveolin-1, anti-phospho-ERK(Tyr204), anti-ERK, anti-phospho-Stat3(Tyr705), and anti-NDRG1 antibodies were from Santa Cruz Biotechnology INC. Anti-phospho-EGFR(Tyr1068) and anti-phospho-ErbB2(Tyr1248) antibodies were from Abcam. Anti-α-tubulin, anti-β-actin, and anti-cholera toxin B subunit antibodies were from Sigma Aldrich. Anti-4EBP1, anti-AMPKα, anti-acetyl-CoA carboxylase, anti-S6 ribosomal protein, anti-p70 S6 kinase, anti-Akt, anti-phospho-4EBP1(Thr37/46), anti-phospho-Akt(Thr308), anti-phospho-Akt(Ser473), anti-phospho-acetyl-CoA carboxylase(Ser79), anti-phospho-AMPKα(Thr172), anti-phospho-p70 S6 kinase(Thr 389), and anti-phospho-S6 Ribosomal protein(Ser240/244) antibodies were from Cell Signaling. Anti-Sestrin2 and anti-REDD1 antibodies were from Proteintech. Cholera toxin subunit B (CT-B) Conjugates were from Molecular Probes. CyTM3 Conjugate was from Jackson ImmunoResarch Lab. EGF was from Perprotech. MEDICA [α,α′-tetramethyl hexadecanedioic acid, HOOC-C(CH3)2-(CH2)12-C(CH3)2-COOH] was synthesized as previously described [17 (link), 26 ].
5
Western Blotting Quantification of S6 Phosphorylation
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Western blotting was performed as described previously44 (link). Fins were lysed in 2 × SDS-sample buffer (125 mM Tris-HCl (pH 6.8), 4% SDS, 20% Glycerol, 0.01% bromophenol blue, 2% 2-mercaptoethanol) containing a protease inhibitor cocktail (Roche, #11836153001) and a phosphatase inhibitor cocktail (Nacalai, #07574-61). Total proteins (5 µg) were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The PVDF membranes were incubated overnight with primary antibodies: anti-γTubulin (Sigma-Aldrich, #T6557) at 1:20000, ribosomal protein S6 Antibody (C-8) (Santa Cruz Biotechnology, #sc-74459) at 1:1000, and anti-phospho-S6 ribosomal protein (Ser240/244) (Cell signaling, #2215) at 1:1000. For detection, horseradish peroxidase-coupled secondary antibodies: goat anti-mouse IgG-HRP at 1:10000 (Santa Cruz Biotechnology, #sc-2055) and mouse anti-rabbit IgG-HRP at 1:10000 (Santa Cruz Biotechnology, #sc-2357), and the ECL Prime Western Blotting Detection Reagent (GE Healthcare, #RPN2232) were used, according to the manufacturer’s instructions. A densitometric analysis of the western blotting was performed using ImageJ, with γTubulin for normalization.
6
Immunolabeling of Autophagy and Proliferation Markers
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The antibodies used were as follows: anti-phospho-S6 ribosomal protein (Ser240/244); anti-LC3-II and anti SQSTM1/P62 (Cell Signaling Technology); antisynaptophysin (R&D Systems); anti Ki67 (clone SP6, Spring Bioscience, Pleasanton, CA, USA). 2nd antibodies are: goat anti rabbit-Cy5; donkey anti rabbit-Cy3; donkey anti goat-Alexa Fluor 488 and donkey anti mouse-Cy3 (Jackson ImmunoResearch). In Situ Cell Death Detection Kit (Roche AG).
RAD001 (everolimus) (LC Laboratories, Woburn, MA, USA) was dissolved first in DMSO, following dilutions with PBS to yield a stock solution of 0.9 mg/mL, which was stored at -20°C. Chloroquine was supplied by Sigma-Aldrich, Israel, and diluted in PBS to solution of 18 mg/mL. CQ solution was prepared freshly every day, whereas RAD001 stock solution aliquots were thawed immediately before treating the animals. The controls were prepared as appropriate, using the same vehicle as for the drug (PBS).
RAD001 (everolimus) (LC Laboratories, Woburn, MA, USA) was dissolved first in DMSO, following dilutions with PBS to yield a stock solution of 0.9 mg/mL, which was stored at -20°C. Chloroquine was supplied by Sigma-Aldrich, Israel, and diluted in PBS to solution of 18 mg/mL. CQ solution was prepared freshly every day, whereas RAD001 stock solution aliquots were thawed immediately before treating the animals. The controls were prepared as appropriate, using the same vehicle as for the drug (PBS).
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