Journal:

Article Title: Multiple Phosphoinositide 3-Kinase-Dependent Steps in Activation of Protein Kinase B

doi: 10.1128/MCB.22.17.6247-6260.2002

Figure Lengend Snippet: Activation of myristoylated ΔPH-PKB-ER. (A) Myr-ΔPH-PKB-ER (200 ng) was expressed in HEK 293 cells for 30 h, followed by serum starvation for 18 h. Cells were treated with 1 μM 4-OHT for the indicated times and lysed in detergent-containing buffer. Myr-ΔPH-PKB-ER was immunoprecipitated with 1 μg of anti-HA antibody, and activity was measured in vitro as described in Materials and Methods. (B) Myr-ΔPH-PKB-ER (200 ng) or A2-ΔPH-PKB-ER (200 ng) was expressed in HEK 293 cells for 30 h, followed by serum starvation for 18 h. Cells were pretreated with LY-294002 (25 μM) where indicated for 15 min, followed by treatment with 4-OHT (1 μM) for 15 min. Cell were lysed and in vitro kinase assays were performed as described above.

Article Snippet: Human epithelial kidney (HEK) 293 cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagles medium (DMEM) medium supplemented with 10% fetal calf serum and antibiotics at 37°C, 5% CO 2 , and humidity.

Techniques: Activation Assay, Immunoprecipitation, Activity Assay, In Vitro

Journal:

Article Title: Multiple Phosphoinositide 3-Kinase-Dependent Steps in Activation of Protein Kinase B

doi: 10.1128/MCB.22.17.6247-6260.2002

Figure Lengend Snippet: Phosphorylation of T308 and S473 of myr-ΔPH-PKB-ER but not A2-ΔPH-PKB-ER. (A) HEK 293 cells were transfected with myr-ΔPH-PKB-ER or A2-ΔPH-PKB-ER (200 ng) or together with myc-PDK-1 (200 ng) where indicated. After 30 h, cells were serum starved for 18 h and then treated where indicated with 4-OHT (1 μM) for 15 min. Cells were lysed, and proteins were separated by SDS-PAGE followed by Western blotting with phospho-specific antibodies raised against PKB T308 or S473. Lysates were also blotted with anti-myc antibody (to detect PDK-1) and anti-HA antibody (to detect total ΔPH-PKB-ER). (B) Wild-type, R474A, and K111A PDK1 or an empty vector blank (lane B) were transfected into HEK 293 cells for 30 h, followed by serum starvation for 18 h. Cells were stimulated with IGF-1 for various times where indicated, and PDK1 was immunoprecipitated with anti-myc 9E10 antibody. PDK1 activity was measured in vitro using recombinant S422D SGK as substrate. After 15 min at 30°C, the reaction was terminated and 32P-labeled SGK was resolved by SDS-PAGE and visualized by autoradiography.

Article Snippet: Human epithelial kidney (HEK) 293 cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagles medium (DMEM) medium supplemented with 10% fetal calf serum and antibiotics at 37°C, 5% CO 2 , and humidity.

Techniques: Transfection, SDS Page, Western Blot, Plasmid Preparation, Immunoprecipitation, Activity Assay, In Vitro, Recombinant, Labeling, Autoradiography

Journal:

Article Title: Multiple Phosphoinositide 3-Kinase-Dependent Steps in Activation of Protein Kinase B

doi: 10.1128/MCB.22.17.6247-6260.2002

Figure Lengend Snippet: (A) PI3K activity is necessary for S473 phosphorylation of myr-ΔPH-PKB-ER. HEK 293 cells were cotransfected with myr-ΔPH-PKB-ER (200 ng) and either empty vector or wild-type myc-PDK-1, myristoylated PDK-1, or myc-R474A-PDK-1 (all at 200 ng) in the wells indicated. Following 30 h to allow expression, cells were serum starved for 18 h and then treated with LY-294002 (25 μM) for 15 min. Cells were then treated with 4-OHT (1 μM) for an additional 15 min, and cells were lysed in ice-cold Triton X-100-containing buffer. Protein lysates were separated by SDS-PAGE and transferred to PVDF membranes, and PKB T308 and S473 phosphorylation was detected as described for Fig. ​Fig.3.3. Lysates were also probed with antibodies to detect total myr-ΔPH-PKB-ER and PDK-1. (B) The catalytic activity of myr-ΔPH-PKB-ER was measured in an in vitro kinase assay following coexpression with empty vector, wild-type PDK1, or myr-PDK1 as described in Materials and Methods. Data are the averages of quadruplicate determinations from two separate experiments, with error bars representing the standard error of the mean. (C) HEK 293 cells were cultured onto glass coverslips and transfected with 1 μg of myr-ΔPH-PKB-ER or A2-ΔPH-PKB-ER. After 24 h, the cells were fixed in 3% formaldehyde and stained with anti-HA antibody, phalloidin, and DAPI (4′,6′-diamidino-2-phenylindole) as described in Materials and Methods. Cells were visualized by confocal microscopy. (D) HEK 293 cells plated on glass coverslips were transfected with 1 μg of PDK1 or 1 μg of Myr-PDK1. After 24 h, the cells were fixed and stained with anti-PDK1 antibody and visualized by confocal microscopy. (E) HEK 293 cells were trasfected with the wild type or Myr-PDK1 (200 ng). After 30 h, cells were serum starved for 18 h and then resuspended in hypotonic lysis buffer. The cytosol (C) and membrane (M) fractions were prepared as described in Materials and Methods. Samples from each were fractionated by SDS-PAGE and immunoblotted simultaneously with anti-PDK1 and anti-PKB antibodies. The myristoylated PDK1 appears at a higher molecular weight than wild-type PDK1 due to hyperphosphorylation (15) (data not shown).

Article Snippet: Human epithelial kidney (HEK) 293 cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagles medium (DMEM) medium supplemented with 10% fetal calf serum and antibiotics at 37°C, 5% CO 2 , and humidity.

Techniques: Activity Assay, Plasmid Preparation, Expressing, SDS Page, In Vitro, Kinase Assay, Cell Culture, Transfection, Staining, Confocal Microscopy, Lysis, Molecular Weight

Journal:

Article Title: Multiple Phosphoinositide 3-Kinase-Dependent Steps in Activation of Protein Kinase B

doi: 10.1128/MCB.22.17.6247-6260.2002

Figure Lengend Snippet: T308 and S473 phosphorylations of myr-ΔPH-PKB-ER are coupled. (A) Wild-type, T308A, or S473A mutant forms of myr-ΔPH-PKB-ER were expressed in 293 cells for 30 h, followed by serum starvation for 18 h. Cells were treated with 4-OHT (1 μM) for 30 min and then lysed in ice-cold Triton X-100-containing buffer. Immunoblotting was performed to detect phosphorylated T308 or S473. (B) HEK 293 cells were transfected with wild-type, T308A, or K179Q myr-ΔPH-PKB-ER as described above. Some cells expressing wild-type myr-ΔPH-PKB-ER were pretreated with 1 μM staurosporin for 30 min prior to treatment for 30 min with 1 μM 4-OHT. Phosphorylated T308 and S473 were detected by immunoblotting. In both panels, total PKB was detected with anti-HA antibody. (C) Cell were prepared as described for panels A and B, and the catalytic activity of the K179Q, S473A, and T308A mutants of myr-ΔPH-PKB-ER were measured in an in vitro kinase assay as described in Materials and Methods. The activation of wild-type (WT) myr-ΔPH-PKB-ER was also determined after a 15-min treatment with various concentrations of staurosporin (STS).

Article Snippet: Human epithelial kidney (HEK) 293 cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagles medium (DMEM) medium supplemented with 10% fetal calf serum and antibiotics at 37°C, 5% CO 2 , and humidity.

Techniques: Mutagenesis, Western Blot, Transfection, Expressing, Activity Assay, In Vitro, Kinase Assay, Activation Assay

Journal:

Article Title: Multiple Phosphoinositide 3-Kinase-Dependent Steps in Activation of Protein Kinase B

doi: 10.1128/MCB.22.17.6247-6260.2002

Figure Lengend Snippet: Translocation of FRB-PKB to membranes leads to PI3K-dependent phosphorylation of S473 and T308 and elevated activity. (A) HEK 293 cells were plated onto glass coverslips and transfected with 300 ng of FRB-PKB and 600 ng of myr-FKBP. After 18 h, the cells were starved of serum for a further 8 h, followed by treatment with AP21967 (200 nM) for 30 min (+) or no treatment (−). Cells were fixed and stained with anti-PKB antibody, anti-phospho-S473 antibody, phalloidin (to visualize polymerized actin), and DAPI where indicated and as described in Materials and Methods, and confocal images were visualized. (B) HEK 293 cells were transiently transfected with myr-FKPB12 (400 ng), FRB-PKB, and GSK-3β (100 ng) where indicated. After 30 h, the cells were starved of serum for 18 h and then treated with LY-294002 (25 μM) for 15 min, followed by AP21967 (200 nM) for the times indicated. Phosphorylation of T308 and S473 and total FRB-PKB were detected by immunoblotting. (C) Activity of FRB-PKB following treatment of cells with AP21967 was determined in an in vitro kinase assay as described in Materials and Methods. The y axis represents the total count per minute of 32P-labeled Crosstide. (D) The in vivo kinase activity of FRB-PKB was verified by cotransfection with GSK-3β and monitoring S9 phosphorylation by using a phospho-specific antibody following a 30-min treatment with AP21967.

Article Snippet: Human epithelial kidney (HEK) 293 cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagles medium (DMEM) medium supplemented with 10% fetal calf serum and antibiotics at 37°C, 5% CO 2 , and humidity.

Techniques: Translocation Assay, Activity Assay, Transfection, Staining, Western Blot, In Vitro, Kinase Assay, Labeling, In Vivo, Cotransfection

Journal:

Article Title: Multiple Phosphoinositide 3-Kinase-Dependent Steps in Activation of Protein Kinase B

doi: 10.1128/MCB.22.17.6247-6260.2002

Figure Lengend Snippet: Effect of staurosporin on FRB-PKB phosphorylation and kinase activity. (A) HEK 293 cells were cotransfected with myr-FKBP (400 ng) and FRB-PKB (200 ng). After 30 h, the cells were starved of serum for 18 h and then treated with 1 μM staurosporin where indicated for 15 min, followed by 200 nM AP21967 for 30 min. Kinase activity was measured, and reserved portions of the cell lysates were immunoblotted with anti-T308 antibody, anti-S473 antibody, and total PKB. (B) HeLa cells transfected with myr-FKBP and FRB-PKB were treated with LY-294002 (25 μM) for 15 min or staurosporin (1 μM) where indicated, followed by 200 nM AP21967 for various times where indicated. Lysates were immunoblotted for T308 and S473 phosphorylation and for total FRB-PKB.

Article Snippet: Human epithelial kidney (HEK) 293 cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagles medium (DMEM) medium supplemented with 10% fetal calf serum and antibiotics at 37°C, 5% CO 2 , and humidity.

Techniques: Activity Assay, Transfection

Journal: iScience

Article Title: Comparison of DNA vaccines with AS03 as an adjuvant and an mRNA vaccine against SARS-CoV-2

doi: 10.1016/j.isci.2023.107120

Figure Lengend Snippet: The recombinant Spike Protein as a DNA-vaccine candidate for SARS-CoV-2 (A) Schematic representation of the prefusion-stabilized SARS-CoV-2 HexaPro ectodomain showing the S1 and S2 subunits. Four additional proline substitutions from the S-2P construct are indicated by the red arrows shown below the construct. SS- Signal sequence; NTD N-terminal domain; RBD- Receptor Binding domain; SD1-2- Subdomain1-2; CH- Central helix; CD-connector domain; HR-heptad repeat FP- fusion peptide. (B) HexaPro Spike protein expressed in Expi293 cells was confirmed by SDS-PAGE. (C–D) His-tagged HexaPro was expressed in Expi293, purified and characterized by SDS-PAGE (right). (E) Expression of HexaPro Spike confirmed by western blot using a commercial anti-His antibody or pooled sera of Spike-immunized mice. (F) Flow cytometric analysis showing the binding of pooled mouse sera of HexaPro immunized mice to the HexaPro Spike expressed on 293T cells. (G–H) The mRNA expression levels of inflammatory cytokines IL-6 and TNFα in HexaPro-transfected cells were detected by qRT-PCR. The bars represent the means with error bars denoting the SD of three samples (∗∗∗significantly different (p < 0.001) by two-tailed unpaired t-test).

Article Snippet: BEAS2B, 293T cells were obtained from American Type Culture Collection (Manassas, VA).

Techniques: Recombinant, Construct, Sequencing, Binding Assay, SDS Page, Purification, Expressing, Western Blot, Transfection, Quantitative RT-PCR, Two Tailed Test

Journal: iScience

Article Title: Comparison of DNA vaccines with AS03 as an adjuvant and an mRNA vaccine against SARS-CoV-2

doi: 10.1016/j.isci.2023.107120

Figure Lengend Snippet:

Article Snippet: BEAS2B, 293T cells were obtained from American Type Culture Collection (Manassas, VA).

Techniques: Virus, Recombinant, Adjuvant, Cell Stimulation, Modification, Expressing, Neutralization, Double-Color Enzymatic ELISPOT, cDNA Synthesis, Transfection, Purification, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Software

Journal:

Article Title: The Long Terminal Repeat of Jaagsiekte Sheep Retrovirus Is Preferentially Active in Differentiated Epithelial Cells of the Lungs

doi:

Figure Lengend Snippet: Cell lines used in this study

Article Snippet: H441 and H358 cells were grown in RPMI 1640 medium (Gibco BRL) adjusted to contain 1.5 g of sodium bicarbonate per liter, 4.5 g of glucose per liter, and 10 mM HEPES with 10% FBS. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Cell line Origin Tissue or cell type ATCC no. or reference A549 Human Lung carcinoma CCL-185 H441 Human BAC HTB-174 H358 Human BAC CRL-5807 293T Human Embryonic kidney Lebkowsky et al. ( 33 ) OA1 Sheep Brain fibroblast CRL-6538 OAT Sheep Sheep testis CRL-6546 FLL Sheep Primary fetal lamb MRI a JS7 Sheep BAC Jassim ( 30 ) CP-MRI Sheep Choroid plexus MRI a CP-ATCC Sheep Choroid plexus CRL-1700 mtCC1-2 Mouse Clara cell Magdaleno et al. ( 34 ) BV2 Mouse Microglia A. Tenner a F9 Mouse Testicular carcinoma CRL-1720 FOP Mouse Mammary carcinoma J. Hassel a IC-21 Mouse Peritoneal macrophage TIB-186 MHS Mouse Alveolar macrophage CRL-2019 C2C12 Mouse Myoblast CRL-1772 ABI-2 Mouse Hybridoma HB-33 MLE-12 Mouse Lung epithelium CRL-2110 TCMK Mouse Mouse kidney CCL-139 NIH 3T3 Mouse Mouse embryo CCL-92 MLE-15 Mouse Type II pneumocyte Wikenheiser et al. ( 59 ) ST3 Mouse Thymus stroma Brightman et al. ( 10 ) Open in a separate window a Cells were provided directly by an investigator or institution with no reference available.

Techniques: