Following fixation, cells were permeabilized by five times washes in 0.1% Triton X-100 in 1X Tris Buffered Saline (TBS). Cells were blocked for 1 hour at RT in Licor Blocking Buffer (Licor), and hybridized overnight at 4°C in primary antibody (1:250 phospho-eIF4E (Ser209) (Abcam), 1:300 phospho-Akt (Ser473) (Cell Signaling), 1:200 phospho-ERK1/2 (Thr202/Tyr204) (Cell Signaling), 1:400 phospho-4EBP1 (Thr70) (Cell Signaling), 1:500 phosphor-P70 (Thr389) (Cell Signaling), 1: 400 phosphor-S6 (Ser235/236) (Cell Signaling), and 1:400 phosphor-mTOR (Ser2448) (Cell Signaling)). Cells were washed 5 times in TBS with 0.1% Tween-20 and hybridized with 1:5000 Cell Tag (Licor) and 1:800 goat anti-mouse 800CW (Licor) or donkey anti-rabbit 800CW (Licor) for 1 hour at RT. Cells were washed 5 times in TBS with 0.1% Tween-20 and a final wash in TBS. Liquid was decanted from the 96 well plate and the plate was scanned using the Odyssey Imager (Licor) and Image Studio (Licor). We compensated for differences in cell numbers by adjusting the phosphorylation levels (800 channel) by the Cell Tag signal (680 channel). Phosphorylation levels are reported as a ratio to the mean signal in the control cells.
Phosphor s6 ser235 236
Manufactured by Cell Signaling Technology
Phosphor-S6 (Ser235/236) is a lab equipment product that detects the phosphorylation of the S6 ribosomal protein at serine residues 235 and 236. This product is used to monitor the activation status of the mTOR signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Donkey anti rabbit 800cw, by LI COR (1 mentions)
Phospho erk1 2 thr202 tyr204, by Cell Signaling Technology (1 mentions)
Goat anti mouse 800cw, by LI COR (1 mentions)
Odyssey imager, by LI COR (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Image studio, by LI COR (1 mentions)
Phosphor mtor ser 2448, by Cell Signaling Technology (1 mentions)
Phospho akt ser473, by Cell Signaling Technology (1 mentions)
Phospho 4e bp1 thr70, by Cell Signaling Technology (1 mentions)
Cell tag, by LI COR (1 mentions)
Cellsens, by Olympus (1 mentions)
Phosphor akt ser473, by Cell Signaling Technology (1 mentions)
2 protocols using phosphor s6 ser235 236
1
Quantitative Phospho-Protein Profiling
2
Evaluating Tumor Microenvironment Biomarkers
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Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tumor sections. Sections were dewaxed, rehydrated, and subjected to antigen retrieval by autoclave incubation in citrate buffer (pH 6.0). The primary antibodies against phosphor-S6 (Ser235/236) and phosphor-AKT (Ser473) were obtained from Cell Signaling Technologies (Danvers, MA) and BrdU from BD Pharmingen (San Jose, CA). After secondary antibody incubation, sections were visualized with a fluorescence method. For detection of the hypoxic region, fluorescein isothiocyanate–labeled hypoxyprobe-1 monoclonal antibody (NPI, Belmont, MA) was used. Apoptosis was detected with the ApopTag Red In Situ Apoptosis Detection Kit (CEMICON, Temecula, CA), and image analysis was performed with software cellSens (Olympus, Tokyo, Japan). The percentage of BrdU-positive cells was measured by Lumina Vision (Mitani Corp., Tokyo, Japan) in two regions of interest: a proximal region (within 50 µm of the pimonidazole-positive zone) and a distal region (more than 50 µm from the pimonidazole-positive zone).
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