Immunohistochemical labeling of embryonic brain sections or dissociated neural cells was performed as described previously (Kim et al., 2006 (link)). The following primary antibodies were used: rabbit anti-mTOR (Cell Signaling), rabbit anti-4EBP1 (Cell Signaling), mouse anti-S6 (Cell Signaling), rabbit anti-TSC2 (Cell Signaling), rabbit anti-phospho-mTOR (Cell Signaling), rabbit anti-phospho-4EBP1 (Cell Signaling), rabbit anti-phospho-S6 (Cell Signaling), rabbit anti-phospho-S6K (Cell Signaling), rabbit anti-phospho-histone H3 (Upstate Biotech), mouse anti-BrdU (Sigma), rabbit anti-Ki67 (Covance), rabbit anti-Sox2 (Chemicon), rabbit anti-Tbr1 (Chemicon), rabbit anti-Cux1 (Santa Cruz), goat anti-Brn1 (Novus Biologicals), rabbit anti-Tbr2 (Abcam), mouse anti-Tuj1 (Sigma). Appropriate secondary antibodies conjugated with Alexa Fluor dyes (Invitrogen) were used to detect primary antibodies.
Rabbit anti phospho s6
Manufactured by Cell Signaling Technology
Sourced in United States
The Rabbit anti-phospho-S6 is a specific antibody that recognizes the phosphorylated form of the ribosomal protein S6. It is a tool used in research applications to detect and monitor the phosphorylation status of S6 protein.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti phospho 4ebp1, by Cell Signaling Technology (3 mentions)
Rabbit anti mtor, by Cell Signaling Technology (2 mentions)
Rabbit anti 4e bp1, by Cell Signaling Technology (2 mentions)
Mouse anti s6, by Cell Signaling Technology (2 mentions)
Rabbit anti tsc2, by Cell Signaling Technology (2 mentions)
Rabbit anti phospho s6k, by Cell Signaling Technology (2 mentions)
Rabbit anti akt, by Cell Signaling Technology (2 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
Mouse anti brdu, by Merck Group (1 mentions)
Rabbit anti sox2, by Merck Group (1 mentions)
Mouse anti tuj1, by Merck Group (1 mentions)
Rabbit anti tbr1, by Merck Group (1 mentions)
Goat anti brn1, by Novus Biologicals (1 mentions)
Rabbit anti phospho mtor, by Cell Signaling Technology (1 mentions)
Rabbit anti cux1, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti tbr2, by Abcam (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Rabbit anti phospho myosin light chain, by Cell Signaling Technology (1 mentions)
Mouse anti neurofilament, by Merck Group (1 mentions)
9 protocols using rabbit anti phospho s6
1
Immunohistochemical Profiling of Neural Cells
2
Imaging subcellular structures in oocytes
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Oocytes were fixed in 4% formaldehyde (Sigma-Aldrich, F8775) in ASW at 16 °C for 1 h under gentle stirring. After fixation, they were washed three times in PBS-Tw (0.1% Tween20 in PBS), permeabilized for 30 min in 0.1% Triton 100-X in PBS and washed three times in PBS-Tw. Oocytes were then incubated in blocking solution (0.1% Tween20, 0.5% BSA in PBS) for 30 min, followed by incubation in the primary antibody in PBS-BSA (1% BSA in PBS) for 24 h at room temperature and three washes in PBS-Tw. Incubation of the secondary antibody and phalloidin (1/200, Alexa FluorTM 488 Phalloidin, Invitrogen, A12379 ) was carried out for 24 h at room temperature. To label the maternal and paternal DNA, Hoechst (Fisher Scientific, 62249) was added at a final concentration of 5 µg ml−1 for the final 10 min of incubation. After incubation, oocytes were washed three times in PBS-Tw and one time in PBS and mounted for imaging. The primary antibodies were mouse anti-neurofilament (1/400, Sigma-Aldrich, N5264, for labelling the myoplasm), rabbit anti-phospho-S6 (1/400, Cell Signaling Technology, 2211, for labelling the endoplasmic reticulum) and rabbit anti-phospho-Myosin light chain (1/500, Cell Signaling Technology, 3674S, for labelling phospho-Myosin). The secondary antibodies were goat anti-mouse/rabbit conjugated to Alexa Fluor 488/546/647 (1/200) Molecular Probes).
3
Western Blot Analysis of Key Proteins
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Western blot analysis was performed following previously established procedures [32] (link). The antibody against human KLF5 was prepared as previously described [14] (link), and those against other proteins are given as follow: rabbit anti-PTEN, rabbit anti–phospho-AKT, rabbit anti–phospho-ERK, rabbit anti-AKT, rabbit anti-ERK, and rabbit anti–phospho-S6 were from Cell Signaling Technology; and rabbit anti–β-actin, rabbit anti-p15, and rabbit anti-mouse Klf5 were from Sigma, Santa Cruz Biotechnology, and Millipore (Temecula, CA), respectively.
4
Western Blot Analysis of Signaling Proteins
5
Molecular Profiling of Telencephalon Development
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Lysates from E13.5 telencephalon were prepared using RIPA buffer and the protein content was determined by a Bio-Rad Protein Assay system. Primary antibodies: rabbit anti-phospho-S6 (Cell Signaling), rabbit anti-phospho-S6K (Cell Signaling), rabbit anti-phospho-4EBP1 (Cell Signaling), rabbit anti-GAPDH (Cell Signaling), rabbit anti-TSC2 (Cell Signaling), rabbit anti-mTOR (Cell Signaling), rabbit anti-phospho-serine (Abcam), mouse anti-GSK3β (BD Transduction Laboratory) or rabbit anti-GSK3β antibody (Upstate). Appropriate secondary antibodies conjugated to HRP were used (Cell Signaling) and the ECL reagents (Amersham) were used for immunodetection.
For quantification of band intensity, blots from three independent experiments for each molecule of interest were used. Signals were measured using ImageJ software and represented by relative intensity versus control. GAPDH was used as an internal control to normalize band intensity.
For quantification of band intensity, blots from three independent experiments for each molecule of interest were used. Signals were measured using ImageJ software and represented by relative intensity versus control. GAPDH was used as an internal control to normalize band intensity.
6
Western Blot Analysis of Cellular Proteins
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Brain and spinal cord sections were homogenized in lysis buffer (10 mM Tris, pH 7.4, 2 mM EDTA, 0.25 M sucrose) supplemented with protease (Sigma) and phosphatase inhibitor cocktails (Thermo Scientific). The lysates were incubated on ice for 30 min, vortexed, and centrifuged at 14.000 g for 10 min at 4°C. Protein concentrations were determined using the BCA assay (Thermo Scientific). Equal amounts of protein (i.e., 20 μg) were mixed with Laemmli loading buffer (Bio-Rad) and loaded on a 10% or 18% SDS-PAGE gel. After separation, proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore), which were incubated with the following primary antibodies overnight: mouse anti-p62/SQSTM1 (1:1000, Abcam), rabbit anti-LC3B (1:1000, Novus biologicals), mouse anti-S6 (1:500, Cell Signaling), rabbit anti-phospho-S6 (1:500, Cell Signaling), mouse anti- β-tubulin (1:1000, Abcam), and rabbit anti-β-actin (1:1000, Abcam). Thereafter, membranes were washed and incubated with the following secondary antibodies for 1 h at RT: anti-rabbit IRDye800CW and anti-mouse IRDye680RD (LI-COR Biosciences). Signals were detected using the Odyssey Imaging System (LI-COR Biosciences) and densitometric values were determined and quantified at non-saturating exposures using the ImageJ software (Schneider et al. 2012 (link)), and normalized against the loading controls.
7
Enterocyte Protein Expression Analysis
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Jejunal IEC from three-month-old wild-type and villin-Cre Hdac1−/−; Hdac2−/− mice were enriched by EDTA treatment. IEC were lysed in 1 X Laemmli buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol) supplemented with protease and phosphatase inhibitors. Protein concentrations were determined with the Pierce BCA Protein Assay kit (Therrmo Fisher Scientific). Amounts of 15 µg of total proteins were separated on 4–12% SDS-polyacrylamide gels, and transferred on PVDF membranes (Roche Molecular Biochemicals). Membranes were incubated 1 h at room temperature or overnight at 4 °C with primary antibodies including rabbit anti-phospho-Stat3 (#9145, Cell Signaling), rabbit anti-Stat3 (#12640, Cell Signaling), rabbit anti-phospho-p38 (#4511, Cell Signaling), rabbit anti-claudin 3 (341700, Invitrogen), rabbit anti-cleaved Notch (#4147, Cell Signaling), rabbit anti-phospho-S6 (#4858, Cell Signaling) and GAPDH (#2118, Cell Signaling). Primary antibodies were recognized with secondary goat anti-rabbit antibodies (Life Technologies) before immune complex detection with Amersham ECLTM Western blotting detection reagents (GE Healthcare, Mississauga, ON, Canada).
8
Immunohistochemical Staining Protocols
9
Quantitative Analysis of Cellular Phosphoproteins
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A total of 20,000 to 30,000 LSK cells were sorted into trichloroacetic acid (TCA), and the volume was adjusted to a final concentration of 10% TCA. Samples were incubated for 15 min on ice and centrifuged at 14,000 revolutions per minute at 4°C for 20 min. Supernatant was carefully removed, and precipitates were washed in prechilled acetone twice. The pellets were air dried, solubilized in NuPAGE LDS Sample Buffer, and heated at 70°C for 10 min. Samples subjected to immunoblot analysis with the indicated antibodies: rabbit anti–phospho-S6 (Cell Signaling Technology, 2215S), rabbit anti–phospho-4E-BP1 (Cell Signaling Technology, 2855S), and mouse anti–β-Actin (Thermo Fisher Scientific, MA5-15739). The immunoreactive bands were detected using horseradish peroxidase–conjugated secondary antibodies (Jackson ImmunoResearch) and enhanced chemiluminescence reagents (Thermo Fisher Scientific). Before reprobing, blots were stripped with stripping buffer [1% SDS and 25 mM glycine (pH 2)].
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