Journal: eLife

Article Title: Ezrin defines TSC complex activation at endosomal compartments through EGFR–AKT signaling

doi: 10.7554/eLife.98523

Figure Lengend Snippet:

Article Snippet: The following primary antibodies were used: rat anti-LAMP-1 (1:400, Santa Cruz sc-19992), mouse anti-LAMP-1 (1:1000, DSHB H4A3), rabbit anti-LAMP1 (1:100, Abcam ab24170), rabbit anti-LC3B (1:200, Novus NB100-2220), rabbit anti-EGF receptor (1:50, Cell Signaling 4267), mouse anti-EEA1 (1:100, BD 610457), rabbit anti-Tuberin/TSC2 (1:100, Cell Signaling 4308), rabbit anti-Hamartin/TSC1 (1:1000, Cell Signaling 6935), mouse anti-EGFR (1:50, Santa Cruz sc-120), chicken anti-GFP (1:500, Abcam ab13970), LysoTracker Red (Invitrogen L7528), CellLight Early Endosomes-RFP (Invitrogen C10587).

Techniques: CRISPR, Transfection, Construct, Sequencing, Recombinant, Purification, Western Blot, Software, Mass Spectrometry, Immunofluorescence, Functional Assay, Fluorescence, Imaging, Staining, Plasmid Preparation

Journal: Endocrinology

Article Title: Activation of the hexosamine pathway leads to phosphorylation of insulin receptor substrate-1 on Ser307 and Ser612 and impairs the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin insulin biosynthetic pathway in RIN pancreatic beta-cells.

doi: 10.1210/en.2003-0939

Figure Lengend Snippet: FIG. 7. Effect of glucosamine on phosphorylation of mTOR and tuberin in RIN -cells. RIN -cells were cultured in serum-free medium for 24 h in the presence or absence of 7.5 mM glucosamine. Cells were then incubated 15 min in the presence or absence of 100 nM insulin. The cells were lysed, and equal amounts of proteins were subjected to SDS-PAGE and immunoblotted with phosphor-specific anti-Ser2248 mTOR (A) or anti-Thr1462 tuberin (B) antibody. To normalize the blots for protein levels, after being immunoblotted with antiphosphospecific antibodies, the blots were stripped and reprobed with anti-mTOR or antituberin antibody. Proteins were detected by using ECL, and band densities were quantified by densitometry. Each bar represents the mean SEM of three independent experiments, and autoradiographs of a representative experiment are shown (*, P 0.05; **, P 0.01).

Article Snippet: The membranes were blotted with anti-Ser473 Akt, anti-Thr389 p70S6 kinase, antiSer2248 mTOR, or anti-Thr1462 tuberin antibody (Cell Signaling).

Techniques: Phospho-proteomics, Cell Culture, Incubation, SDS Page

Journal:

Article Title: The coordinate regulation of the p53 and mTOR pathways in cells

doi: 10.1073/pnas.0502857102

Figure Lengend Snippet: mTOR inhibition by p53 in MEFs requires both TSC1 and TSC2. (A) TSC1-/- MEFs. (B) WTor TSC2-/- p53-/- MEFs, as indicated, were treated with 20 μM etoposide for the indicated amount of time. (C) TSC2-/- p53-/- MEFs were transiently transfected with either p53 expression vector or empty control vector as indicated. Twenty-four hours posttransfection, the cells were treated with 20 μM etoposide as indicated or left untreated for 8 h (lanes 3 and 4) or 12 h (lanes 1, 2, 5, and 6). Cells from A, B, and C were collected, and the levels of phosphorylated p70S6K (T389), p53, and actin were determined by Western blot analyses.

Article Snippet: Assay-on-demand for human tuberin ( TSC2 ) (catalog no. Hs00241068_s1), human PTEN (catalog no. Hs00829813_m1), human actin (catalog no. Hs99999903_m1) were purchased from Applied Biosystems.

Techniques: Inhibition, Transfection, Expressing, Plasmid Preparation, Western Blot

Journal:

Article Title: The coordinate regulation of the p53 and mTOR pathways in cells

doi: 10.1073/pnas.0502857102

Figure Lengend Snippet: The mRNA and protein levels of PTEN and TSC2 are up-regulated upon p53 activation in V138 cells. (A and C) V138 cells and the parental H1299 cells were cultured at 37°C for 24 h before being shifted to 32°C for the indicated amount of time. Cells were collected, and total RNA were prepared. The mRNA levels of TSC2 (A) and PTEN (C) were determined by real-time PCR and normalized against the mRNA levels of actin. (B and D) V138 cells were cultured at 37°C for 24 h before being shifted to 32°C for the indicated amount of time. Cells were collected, and the protein levels of TSC2 (B) and PTEN (D) were detected by Western blot analyses. The levels of protein expression were quantified by densitometry and normalized against those of actin.

Article Snippet: Assay-on-demand for human tuberin ( TSC2 ) (catalog no. Hs00241068_s1), human PTEN (catalog no. Hs00829813_m1), human actin (catalog no. Hs99999903_m1) were purchased from Applied Biosystems.

Techniques: Activation Assay, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Expressing

Journal: iScience

Article Title: MTOR modulation induces selective perturbations in histone methylation which influence the anti-proliferative effects of mTOR inhibitors

doi: 10.1016/j.isci.2024.109188

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Article Snippet: Rabbit monoclonal Tuberin/TSC2 (D93F12) XP® antibody, 1:1000 dilution , Cell Signaling Technologies , Cat#4308; RRID: AB_10547134.

Techniques: Marker, Recombinant, Protease Inhibitor, Picogreen Assay, Extraction, Suspension, cDNA Synthesis, RNA Extraction, shRNA, Control, Software