p s6 Search Results


anti phos s6k1  (Santa Cruz Biotechnology)
95
Santa Cruz Biotechnology anti phos s6k1
Anti Phos S6k1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti p rps6  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology anti p rps6
Anti P Rps6, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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alphalisa surefire assay kit  (Revvity)
90
Revvity alphalisa surefire assay kit
Alphalisa Surefire Assay Kit, supplied by Revvity, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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p70s6k1  (Novus Biologicals)
91
Novus Biologicals p70s6k1
Aβ facilitates HIF1α synthesis and autophagy inhibition via mTOR activation. (A) SK-N-MC cells were exposed to Aβ (5 μM) for 0–48 h. HIF1α and β-actin expression was analyzed by western blot. n = 3. (B) Cells were pretreated with NAC (1 mM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression were analyzed by western blot. n = 3. (C,E) Cells were incubated with rapamycin (10 nM) for 30 min prior to Aβ treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) and 4EBP1, phosphorylation of <t>p70S6K1</t> (Thr 389), HIF1α and β-actin were analyzed by western blot. n = 6. (D) Protein samples were immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples were blotted with 4EBP1 and eIF4E-specific antibodies. n = 3. (F) Cells were exposed to PF4708671 (10 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression was detected by western blot. n = 6. (G) Cells were exposed to cycloheximide (4 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expressions were detected by western blot. n = 6. (H) Cells were pretreated with rapamycin (10 nM) for 30 min, incubated with Aβ for 24 h and analyzed by western blotting with LC3, p62 and β-actin specific antibodies. n = 3–6. (I) LC3 puncta was visualized by confocal microscopy. Presented results are merged images. Green and red fluorescents indicate LC3 and PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells were pretreated with trehalose (10 μM) for 30 min prior to Aβ treatment for 24 h. Cytotoxicity was measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± SE. n = 6. Each blot image was presented as representative image. * p < 0.05 vs. control, # p < 0.05 vs. Aβ treatment.
P70s6k1, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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capsaicin powder  (Chem Impex International)
95
Chem Impex International capsaicin powder
Aβ facilitates HIF1α synthesis and autophagy inhibition via mTOR activation. (A) SK-N-MC cells were exposed to Aβ (5 μM) for 0–48 h. HIF1α and β-actin expression was analyzed by western blot. n = 3. (B) Cells were pretreated with NAC (1 mM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression were analyzed by western blot. n = 3. (C,E) Cells were incubated with rapamycin (10 nM) for 30 min prior to Aβ treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) and 4EBP1, phosphorylation of <t>p70S6K1</t> (Thr 389), HIF1α and β-actin were analyzed by western blot. n = 6. (D) Protein samples were immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples were blotted with 4EBP1 and eIF4E-specific antibodies. n = 3. (F) Cells were exposed to PF4708671 (10 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression was detected by western blot. n = 6. (G) Cells were exposed to cycloheximide (4 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expressions were detected by western blot. n = 6. (H) Cells were pretreated with rapamycin (10 nM) for 30 min, incubated with Aβ for 24 h and analyzed by western blotting with LC3, p62 and β-actin specific antibodies. n = 3–6. (I) LC3 puncta was visualized by confocal microscopy. Presented results are merged images. Green and red fluorescents indicate LC3 and PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells were pretreated with trehalose (10 μM) for 30 min prior to Aβ treatment for 24 h. Cytotoxicity was measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± SE. n = 6. Each blot image was presented as representative image. * p < 0.05 vs. control, # p < 0.05 vs. Aβ treatment.
Capsaicin Powder, supplied by Chem Impex International, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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amoxicillin clavulanate potassium amc  (Valiant Co Ltd)
96
Valiant Co Ltd amoxicillin clavulanate potassium amc
Aβ facilitates HIF1α synthesis and autophagy inhibition via mTOR activation. (A) SK-N-MC cells were exposed to Aβ (5 μM) for 0–48 h. HIF1α and β-actin expression was analyzed by western blot. n = 3. (B) Cells were pretreated with NAC (1 mM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression were analyzed by western blot. n = 3. (C,E) Cells were incubated with rapamycin (10 nM) for 30 min prior to Aβ treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) and 4EBP1, phosphorylation of <t>p70S6K1</t> (Thr 389), HIF1α and β-actin were analyzed by western blot. n = 6. (D) Protein samples were immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples were blotted with 4EBP1 and eIF4E-specific antibodies. n = 3. (F) Cells were exposed to PF4708671 (10 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression was detected by western blot. n = 6. (G) Cells were exposed to cycloheximide (4 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expressions were detected by western blot. n = 6. (H) Cells were pretreated with rapamycin (10 nM) for 30 min, incubated with Aβ for 24 h and analyzed by western blotting with LC3, p62 and β-actin specific antibodies. n = 3–6. (I) LC3 puncta was visualized by confocal microscopy. Presented results are merged images. Green and red fluorescents indicate LC3 and PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells were pretreated with trehalose (10 μM) for 30 min prior to Aβ treatment for 24 h. Cytotoxicity was measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± SE. n = 6. Each blot image was presented as representative image. * p < 0.05 vs. control, # p < 0.05 vs. Aβ treatment.
Amoxicillin Clavulanate Potassium Amc, supplied by Valiant Co Ltd, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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amoxicillin trihydrate  (LGC Standards)
93
LGC Standards amoxicillin trihydrate
Aβ facilitates HIF1α synthesis and autophagy inhibition via mTOR activation. (A) SK-N-MC cells were exposed to Aβ (5 μM) for 0–48 h. HIF1α and β-actin expression was analyzed by western blot. n = 3. (B) Cells were pretreated with NAC (1 mM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression were analyzed by western blot. n = 3. (C,E) Cells were incubated with rapamycin (10 nM) for 30 min prior to Aβ treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) and 4EBP1, phosphorylation of <t>p70S6K1</t> (Thr 389), HIF1α and β-actin were analyzed by western blot. n = 6. (D) Protein samples were immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples were blotted with 4EBP1 and eIF4E-specific antibodies. n = 3. (F) Cells were exposed to PF4708671 (10 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression was detected by western blot. n = 6. (G) Cells were exposed to cycloheximide (4 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expressions were detected by western blot. n = 6. (H) Cells were pretreated with rapamycin (10 nM) for 30 min, incubated with Aβ for 24 h and analyzed by western blotting with LC3, p62 and β-actin specific antibodies. n = 3–6. (I) LC3 puncta was visualized by confocal microscopy. Presented results are merged images. Green and red fluorescents indicate LC3 and PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells were pretreated with trehalose (10 μM) for 30 min prior to Aβ treatment for 24 h. Cytotoxicity was measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± SE. n = 6. Each blot image was presented as representative image. * p < 0.05 vs. control, # p < 0.05 vs. Aβ treatment.
Amoxicillin Trihydrate, supplied by LGC Standards, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ampicillin sodium salt  (Chem Impex International)
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Chem Impex International ampicillin sodium salt
Aβ facilitates HIF1α synthesis and autophagy inhibition via mTOR activation. (A) SK-N-MC cells were exposed to Aβ (5 μM) for 0–48 h. HIF1α and β-actin expression was analyzed by western blot. n = 3. (B) Cells were pretreated with NAC (1 mM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression were analyzed by western blot. n = 3. (C,E) Cells were incubated with rapamycin (10 nM) for 30 min prior to Aβ treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) and 4EBP1, phosphorylation of <t>p70S6K1</t> (Thr 389), HIF1α and β-actin were analyzed by western blot. n = 6. (D) Protein samples were immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples were blotted with 4EBP1 and eIF4E-specific antibodies. n = 3. (F) Cells were exposed to PF4708671 (10 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression was detected by western blot. n = 6. (G) Cells were exposed to cycloheximide (4 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expressions were detected by western blot. n = 6. (H) Cells were pretreated with rapamycin (10 nM) for 30 min, incubated with Aβ for 24 h and analyzed by western blotting with LC3, p62 and β-actin specific antibodies. n = 3–6. (I) LC3 puncta was visualized by confocal microscopy. Presented results are merged images. Green and red fluorescents indicate LC3 and PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells were pretreated with trehalose (10 μM) for 30 min prior to Aβ treatment for 24 h. Cytotoxicity was measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± SE. n = 6. Each blot image was presented as representative image. * p < 0.05 vs. control, # p < 0.05 vs. Aβ treatment.
Ampicillin Sodium Salt, supplied by Chem Impex International, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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s-6 (1) mather, m. l.; rides, m.; allen, c. r. g.; tomlins, p. e  (Meso Scale Diagnostics LLC)
90
Meso Scale Diagnostics LLC s-6 (1) mather, m. l.; rides, m.; allen, c. r. g.; tomlins, p. e
Aβ facilitates HIF1α synthesis and autophagy inhibition via mTOR activation. (A) SK-N-MC cells were exposed to Aβ (5 μM) for 0–48 h. HIF1α and β-actin expression was analyzed by western blot. n = 3. (B) Cells were pretreated with NAC (1 mM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression were analyzed by western blot. n = 3. (C,E) Cells were incubated with rapamycin (10 nM) for 30 min prior to Aβ treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) and 4EBP1, phosphorylation of <t>p70S6K1</t> (Thr 389), HIF1α and β-actin were analyzed by western blot. n = 6. (D) Protein samples were immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples were blotted with 4EBP1 and eIF4E-specific antibodies. n = 3. (F) Cells were exposed to PF4708671 (10 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression was detected by western blot. n = 6. (G) Cells were exposed to cycloheximide (4 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expressions were detected by western blot. n = 6. (H) Cells were pretreated with rapamycin (10 nM) for 30 min, incubated with Aβ for 24 h and analyzed by western blotting with LC3, p62 and β-actin specific antibodies. n = 3–6. (I) LC3 puncta was visualized by confocal microscopy. Presented results are merged images. Green and red fluorescents indicate LC3 and PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells were pretreated with trehalose (10 μM) for 30 min prior to Aβ treatment for 24 h. Cytotoxicity was measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± SE. n = 6. Each blot image was presented as representative image. * p < 0.05 vs. control, # p < 0.05 vs. Aβ treatment.
S 6 (1) Mather, M. L.; Rides, M.; Allen, C. R. G.; Tomlins, P. E, supplied by Meso Scale Diagnostics LLC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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p-s6 ≤10  (CH Instruments)
90
CH Instruments p-s6 ≤10
Aβ facilitates HIF1α synthesis and autophagy inhibition via mTOR activation. (A) SK-N-MC cells were exposed to Aβ (5 μM) for 0–48 h. HIF1α and β-actin expression was analyzed by western blot. n = 3. (B) Cells were pretreated with NAC (1 mM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression were analyzed by western blot. n = 3. (C,E) Cells were incubated with rapamycin (10 nM) for 30 min prior to Aβ treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) and 4EBP1, phosphorylation of <t>p70S6K1</t> (Thr 389), HIF1α and β-actin were analyzed by western blot. n = 6. (D) Protein samples were immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples were blotted with 4EBP1 and eIF4E-specific antibodies. n = 3. (F) Cells were exposed to PF4708671 (10 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression was detected by western blot. n = 6. (G) Cells were exposed to cycloheximide (4 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expressions were detected by western blot. n = 6. (H) Cells were pretreated with rapamycin (10 nM) for 30 min, incubated with Aβ for 24 h and analyzed by western blotting with LC3, p62 and β-actin specific antibodies. n = 3–6. (I) LC3 puncta was visualized by confocal microscopy. Presented results are merged images. Green and red fluorescents indicate LC3 and PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells were pretreated with trehalose (10 μM) for 30 min prior to Aβ treatment for 24 h. Cytotoxicity was measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± SE. n = 6. Each blot image was presented as representative image. * p < 0.05 vs. control, # p < 0.05 vs. Aβ treatment.
P S6 ≤10, supplied by CH Instruments, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/p+s6/p+s6++10/pm38673889-100-7-8
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p-s6  (CH Instruments)
90
CH Instruments p-s6
Aβ facilitates HIF1α synthesis and autophagy inhibition via mTOR activation. (A) SK-N-MC cells were exposed to Aβ (5 μM) for 0–48 h. HIF1α and β-actin expression was analyzed by western blot. n = 3. (B) Cells were pretreated with NAC (1 mM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression were analyzed by western blot. n = 3. (C,E) Cells were incubated with rapamycin (10 nM) for 30 min prior to Aβ treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) and 4EBP1, phosphorylation of <t>p70S6K1</t> (Thr 389), HIF1α and β-actin were analyzed by western blot. n = 6. (D) Protein samples were immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples were blotted with 4EBP1 and eIF4E-specific antibodies. n = 3. (F) Cells were exposed to PF4708671 (10 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression was detected by western blot. n = 6. (G) Cells were exposed to cycloheximide (4 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expressions were detected by western blot. n = 6. (H) Cells were pretreated with rapamycin (10 nM) for 30 min, incubated with Aβ for 24 h and analyzed by western blotting with LC3, p62 and β-actin specific antibodies. n = 3–6. (I) LC3 puncta was visualized by confocal microscopy. Presented results are merged images. Green and red fluorescents indicate LC3 and PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells were pretreated with trehalose (10 μM) for 30 min prior to Aβ treatment for 24 h. Cytotoxicity was measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± SE. n = 6. Each blot image was presented as representative image. * p < 0.05 vs. control, # p < 0.05 vs. Aβ treatment.
P S6, supplied by CH Instruments, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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ps6 (n7-548  (Becton Dickinson)
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Becton Dickinson ps6 (n7-548
A: HMC-1.1 cells (upper panel), HMC-1.2 cells (middle panel), and KU812 cells (lower panel) were incubated with control medium (Co) or medium containing NVP-BEZ235 (1 or 10 µM) or RAD001 (1 or 10 µM) at 37°C for 4 hours. Thereafter, cells were permeabilized and stained with antibodies against pAkt (S473), <t>pS6,</t> or pSTAT5 as described in the text. Expression of phosphorylated (p) signaling molecules in HMC-1 cells and KU812 cells were determined by flow cytometry. Results show the staining index (mean fluorescence intensity values corrected for the isotype) and represent the mean±S.D. from three independent experiments. B: HMC-1.1 cells (upper panel), HMC-1.2 cells (middle panel), and KU812 cells (lower panel) were cultured in the absence or presence of NVP-BEZ235 (1 µM) RAD001 (1 µM) at 37°C for 4 hours. Thereafter, cells were lysed and Western blotting was performed using antibodies against pAkt (S473), Akt, pSTAT5, STAT5, pS6 and β-Actin. C: Western blot results were quantified by densitometry in HMC-1.1 cells (upper panel), HMC-1.2 cells (middle panel), and KU812 cells (lower panel). Results represent the mean±S.D. from three independent experiments.
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Image Search Results


Aβ facilitates HIF1α synthesis and autophagy inhibition via mTOR activation. (A) SK-N-MC cells were exposed to Aβ (5 μM) for 0–48 h. HIF1α and β-actin expression was analyzed by western blot. n = 3. (B) Cells were pretreated with NAC (1 mM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression were analyzed by western blot. n = 3. (C,E) Cells were incubated with rapamycin (10 nM) for 30 min prior to Aβ treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) and 4EBP1, phosphorylation of p70S6K1 (Thr 389), HIF1α and β-actin were analyzed by western blot. n = 6. (D) Protein samples were immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples were blotted with 4EBP1 and eIF4E-specific antibodies. n = 3. (F) Cells were exposed to PF4708671 (10 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression was detected by western blot. n = 6. (G) Cells were exposed to cycloheximide (4 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expressions were detected by western blot. n = 6. (H) Cells were pretreated with rapamycin (10 nM) for 30 min, incubated with Aβ for 24 h and analyzed by western blotting with LC3, p62 and β-actin specific antibodies. n = 3–6. (I) LC3 puncta was visualized by confocal microscopy. Presented results are merged images. Green and red fluorescents indicate LC3 and PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells were pretreated with trehalose (10 μM) for 30 min prior to Aβ treatment for 24 h. Cytotoxicity was measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± SE. n = 6. Each blot image was presented as representative image. * p < 0.05 vs. control, # p < 0.05 vs. Aβ treatment.

Journal: Frontiers in Molecular Neuroscience

Article Title: Amyloid β1-42 (Aβ1-42) Induces the CDK2-Mediated Phosphorylation of Tau through the Activation of the mTORC1 Signaling Pathway While Promoting Neuronal Cell Death

doi: 10.3389/fnmol.2017.00229

Figure Lengend Snippet: Aβ facilitates HIF1α synthesis and autophagy inhibition via mTOR activation. (A) SK-N-MC cells were exposed to Aβ (5 μM) for 0–48 h. HIF1α and β-actin expression was analyzed by western blot. n = 3. (B) Cells were pretreated with NAC (1 mM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression were analyzed by western blot. n = 3. (C,E) Cells were incubated with rapamycin (10 nM) for 30 min prior to Aβ treatment for 24 h. Phosphorylation of 4EBP1 (Thr 37/46) and 4EBP1, phosphorylation of p70S6K1 (Thr 389), HIF1α and β-actin were analyzed by western blot. n = 6. (D) Protein samples were immunoprecipitated by eukaryotic translation initiation factor 4E (eIF4E) antibody-conjugated protein A/G agarose beads. Samples were blotted with 4EBP1 and eIF4E-specific antibodies. n = 3. (F) Cells were exposed to PF4708671 (10 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expression was detected by western blot. n = 6. (G) Cells were exposed to cycloheximide (4 μM) for 30 min prior to Aβ treatment for 24 h. HIF1α and β-actin expressions were detected by western blot. n = 6. (H) Cells were pretreated with rapamycin (10 nM) for 30 min, incubated with Aβ for 24 h and analyzed by western blotting with LC3, p62 and β-actin specific antibodies. n = 3–6. (I) LC3 puncta was visualized by confocal microscopy. Presented results are merged images. Green and red fluorescents indicate LC3 and PI respectively. Scale bars, 50 μm (magnification × 600). (J) Cells were pretreated with trehalose (10 μM) for 30 min prior to Aβ treatment for 24 h. Cytotoxicity was measured by MTT assay at an absorbance of 545 nm using a microplate reader. Data present the mean ± SE. n = 6. (K) Cell viability was measured by trypan blue exclusion assay. Data are presented as a mean ± SE. n = 6. Each blot image was presented as representative image. * p < 0.05 vs. control, # p < 0.05 vs. Aβ treatment.

Article Snippet: The antibodies of hypoxia inducible factor (HIF1α; NB100-105), p70S6K1 (NB600-1049), LC3 (NB100-2220) and p62 (NBP1-48320) were obtained from Novus Biologicals (Littleton, CO, USA) and the HRP-conjugated goat anti-rabbit IgG was purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Activation Assay, Expressing, Western Blot, Incubation, Phospho-proteomics, Immunoprecipitation, Confocal Microscopy, MTT Assay, Trypan Blue Exclusion Assay, Control

A: HMC-1.1 cells (upper panel), HMC-1.2 cells (middle panel), and KU812 cells (lower panel) were incubated with control medium (Co) or medium containing NVP-BEZ235 (1 or 10 µM) or RAD001 (1 or 10 µM) at 37°C for 4 hours. Thereafter, cells were permeabilized and stained with antibodies against pAkt (S473), pS6, or pSTAT5 as described in the text. Expression of phosphorylated (p) signaling molecules in HMC-1 cells and KU812 cells were determined by flow cytometry. Results show the staining index (mean fluorescence intensity values corrected for the isotype) and represent the mean±S.D. from three independent experiments. B: HMC-1.1 cells (upper panel), HMC-1.2 cells (middle panel), and KU812 cells (lower panel) were cultured in the absence or presence of NVP-BEZ235 (1 µM) RAD001 (1 µM) at 37°C for 4 hours. Thereafter, cells were lysed and Western blotting was performed using antibodies against pAkt (S473), Akt, pSTAT5, STAT5, pS6 and β-Actin. C: Western blot results were quantified by densitometry in HMC-1.1 cells (upper panel), HMC-1.2 cells (middle panel), and KU812 cells (lower panel). Results represent the mean±S.D. from three independent experiments.

Journal: PLoS ONE

Article Title: The PI3-Kinase/mTOR-Targeting Drug NVP-BEZ235 Inhibits Growth and IgE-Dependent Activation of Human Mast Cells and Basophils

doi: 10.1371/journal.pone.0029925

Figure Lengend Snippet: A: HMC-1.1 cells (upper panel), HMC-1.2 cells (middle panel), and KU812 cells (lower panel) were incubated with control medium (Co) or medium containing NVP-BEZ235 (1 or 10 µM) or RAD001 (1 or 10 µM) at 37°C for 4 hours. Thereafter, cells were permeabilized and stained with antibodies against pAkt (S473), pS6, or pSTAT5 as described in the text. Expression of phosphorylated (p) signaling molecules in HMC-1 cells and KU812 cells were determined by flow cytometry. Results show the staining index (mean fluorescence intensity values corrected for the isotype) and represent the mean±S.D. from three independent experiments. B: HMC-1.1 cells (upper panel), HMC-1.2 cells (middle panel), and KU812 cells (lower panel) were cultured in the absence or presence of NVP-BEZ235 (1 µM) RAD001 (1 µM) at 37°C for 4 hours. Thereafter, cells were lysed and Western blotting was performed using antibodies against pAkt (S473), Akt, pSTAT5, STAT5, pS6 and β-Actin. C: Western blot results were quantified by densitometry in HMC-1.1 cells (upper panel), HMC-1.2 cells (middle panel), and KU812 cells (lower panel). Results represent the mean±S.D. from three independent experiments.

Article Snippet: For staining of cytoplasmic molecules, KU812 and HMC-1 cells were permeabilized by methanol (−20°C, 15 minutes) and then incubated with mAb against phosphorylated Akt (pAkt, S473) (M89-61), pS6 (N7-548), pSTAT5 (clone 47), or activated caspase 3 (C92-605) (all from Becton Dickinson Biosciences) for 30 minutes.

Techniques: Incubation, Staining, Expressing, Flow Cytometry, Fluorescence, Cell Culture, Western Blot