antihuman mouse akt  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: Survival of salivary gland cancer stem cells requires mTOR signaling

doi: 10.1038/s41419-021-03391-7

Figure Lengend Snippet: a Western blots for AKT, p-S473-AKT, mTOR, p-S2448-mTOR, S6K1, p-T421/S424-S6K1, Bmi-1, and B-actin after exposure of bulk MEC tumor cells (UM-HMC-1, UM-HMC-3A, and UM-HMC-3B) to increasing concentrations of AKT inhibitors (0.2–20 μM buparlisib or LY2940002) or vehicle control. Graphs depict the fraction of ALDH high CD44 high cells identified by flow cytometry in MEC tumor cells (UM-HMC-1, UM-HMC-3A, and UM-HMC-3B) after exposure to increasing concentrations of buparlisib, LY2940002, or vehicle control. b Western blots for AKT, p-S473-AKT, mTOR, p-S2448-mTOR, S6K1, p-T421/S424-S6K1, and Bmi-1 after exposure of bulk MEC tumor cells to increasing concentrations of mTOR inhibitors (0.2–20 ng/ml rapamycin or temsirolimus) or vehicle control. Graphs depict the fraction of ALDH high CD44 high cells identified by flow cytometry in MEC tumor cells after exposure to increasing concentrations of rapamycin or temsirolimus. c Western blots for AKT, p-S473-AKT, mTOR, p-S2448-mTOR, S6K1, p-T421/S424-S6K1, and Bmi-1 after exposure of bulk MEC tumor cells to increasing concentrations of S6K1 inhibitor (2–200 nM PF4708671) or vehicle control. Graphs depict the fraction of ALDH high CD44 high cells identified by flow cytometry in UM-HMC-1, UM-HMC-3A, and UM-HMC-3B cell lines after exposure to increasing concentrations of the S6K1 inhibitor. d Western blots for mTOR, p-S2448-mTOR, S6K1, p-T421/S424-S6K1, 4E-BP1, and p-S65-4E-BP1 upon exposure of UM-HMC-3A cells to increasing concentrations of temsirolimus for 24 h. e Western blots for mTOR, p-S2448-mTOR, Rictor, and p-T1135-Rictor upon exposure of UM-HMC-3A cells to increasing concentrations of temsirolimus for 24 h. Asterisk depicts p < 0.05, as determined by one-way ANOVA followed by post-hoc tests for multiple comparisons between vehicle-treated and cells that were exposed to experimental compounds. Error bars indicate standard deviation (SD). Graphs for impacts of treatment on the fraction of ALDH high CD44 high cells depict data from four wells per experimental condition. Experiments were performed three independent times to verify reproducibility of the data.

Article Snippet: Membranes were incubated overnight at 4 °C with 1:1000 dilution of the following antibodies: rabbit antihuman p-AKT Ser 473 (Cell Signaling, Danvers, MA, USA), mouse antihuman AKT (Cell Signaling), rabbit antihuman p-Rictor Thr1135 (Cell Signaling), mouse antihuman Rictor (Santa Cruz Biotechnology; Dallas, TX, USA), rabbit antihuman p-mTOR Ser 2448 (Santa Cruz), rabbit antihuman mTOR (Cell Signaling), mouse antihuman p-4E-BP1 Ser 65 (Santa Cruz), mouse antihuman p-4E-BP1 (Santa Cruz), rabbit antihuman p-S6K1 Thr 421/Ser 424 (Santa Cruz), rabbit antihuman S6K1 (Cell Signaling), rabbit antihuman Bmi-1 (Cell Signaling).

Techniques: Western Blot, Control, Flow Cytometry, Standard Deviation

Journal: Chest

Article Title: IL-27 Is Elevated in Patients With COPD and Patients With Pulmonary TB and Induces Human Bronchial Epithelial Cells to Produce CXCL10

doi: 10.1378/chest.10-3297

Figure Lengend Snippet: Effects of IL-27 on the activation of the PI3K-Akt signaling pathway in PBEC grown on air-liquid interface culture. PBEC were incubated with or without IL-27 (100 ng/mL) for 5 min. A, Intracellular expression of phosphorylated PI3K. B, Intracellular expression of phosphorylated Akt. The isotypic control represents the cell populations stained with antimouse IgG1 isotype control. C, Activation of PI3K was determined by enzyme-linked immunosorbent assay 15 min after stimulation with IL-27. *** P < .001 vs medium control. D, Phosphorylated Akt and total Akt levels were determined by Western blot analysis 15 min after stimulation with IL-27. The results are from three independent experiments. PI3K = phosphatidylinositol 3-OH kinase. See , legends for expansion of abbreviations.

Article Snippet: For flow cytometric analysis, permeabilized PBEC were stained with mouse antihuman phosphorylated Akt (BD Pharmingen) and phosphorylated PI3K (Cell Signaling Technology) or mouse IgG1 antibodies (BD Pharmingen), followed by fluorescein isothiocyanate-conjugated goat antimouse secondary antibodies.

Techniques: Activation Assay, Incubation, Expressing, Staining, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Chest

Article Title: IL-27 Is Elevated in Patients With COPD and Patients With Pulmonary TB and Induces Human Bronchial Epithelial Cells to Produce CXCL10

doi: 10.1378/chest.10-3297

Figure Lengend Snippet: Effects of siRNA-PI3K p110α, siRNA-PI3K p110β, and siRNA-Akt on IL-27-induced CXCL10 expression. BEAS-2B cells were transfected with or without siRNA-PI3K p110α and siRNA-PI3K p110β, and their blocking effects were validated by Western blot. A, Blocking effects on the expression of PI3K p110α. B, Blocking effects on the expression of PI3K p110β. C, Blocking effects on the phosphorylation of Akt. BEAS-2B cells were transfected with siRNA-PI3K p110α, siRNA-PI3K p110β, and siRNA-Akt. The cells and cell supernatants were then harvested at 2 h and 24 h, respectively, after stimulation with 100 ng/mL of IL-27. D, Level of CXCL10 gene expression measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). E, Level of protein production measured by RT-PCR and ELISA. Results are expressed as the arithmetic mean ± SD from three independent experiments. * P < .05, ** P < .01, and *** P < .001 when compared between groups denoted by horizontal lines. GADPH = glyceraldehyde 3-phosphate dehydrogenase. See , legends for expansion of other abbreviations.

Article Snippet: For flow cytometric analysis, permeabilized PBEC were stained with mouse antihuman phosphorylated Akt (BD Pharmingen) and phosphorylated PI3K (Cell Signaling Technology) or mouse IgG1 antibodies (BD Pharmingen), followed by fluorescein isothiocyanate-conjugated goat antimouse secondary antibodies.

Techniques: Expressing, Transfection, Blocking Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay