Journal: Immunology

Article Title: NKG2A ‐checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA‐E

doi: 10.1111/imm.13515

Figure Lengend Snippet: Use of a reporter cell system to evaluate NKG2A/CD94 inhibition and its interaction with peptide‐HLA‐E complexes. (a) Schematic showing the interaction between NKG2A and the TCR/CD3 complex on the Jurkat reporter cells with HLA‐E‐peptide complexes and membrane‐bound anti‐CD3‐scFv on the T cell stimulator cells (K562S). (b) Surface expression of NKG2A on control and NKG2A/CD94 expressing reporter cells. (c) Schematic representation of a construct harbouring the sequences of the H1N1 leader, a 9‐mer peptide, GS‐linker (#1), β 2 ‐microglobulin, GS‐linker (#2) and HLA‐E*0103. In the table, the five different 9‐mer peptides used in this study are depicted. Flow cytometric analysis of T cell stimulator cells (K562S RFP). Left panel; HLA‐E expression on control K562S cells (open histogram) and K562S transduced to express peptide‐HLA‐E complexes (filled histograms). Right panel; expression of membrane‐bound anti‐CD3 Ab fragment (CD14‐stem) on K562 cells (open histogram) and K562S (with and without HLA‐E) cells (filled histograms). (d) Gating strategy of one representative experiment of control reporter cells and NKG2A/CD94 reporter cells stimulated with control K562S and K562S‐HLA‐E HLA‐G , K562S‐HLA‐E HLA‐B8 , K562S‐HLA‐E HLA‐A2 , K562S‐HLA‐E HLA‐C4 and K562S‐HLA‐E hsp60 . Open histogram: unstimulated cells. NFκB‐eCFP expression was measured via flow cytometry. (e) Control and NKG2A/CD94 expressing reporter cells were stimulated with the indicated K562S cells. Reporter activation is shown as the geometric mean of fluorescence intensity (gMFI) of NFκB activation. Lower panel: reporter activation is shown as fold induction (gMFI of K562S‐HLA‐E peptide ‐stimulated cells/gMFI control K562S‐stimulated cells). Results of seven independent experiments performed in duplicates are depicted. For statistical evaluation, a two‐way ANOVA followed by Bonferroni post hoc test was performed (*** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; ns p > 0.05).

Article Snippet: Human NKG2A antibody (clone REA110) was from Miltenyi Biotec and clone Z199 was from Beckman Coulter Inc. Human NKG2A antibody (131411) and NKG2C antibody (134522) were from R&D systems.

Techniques: Inhibition, Membrane, Expressing, Control, Construct, Flow Cytometry, Activation Assay, Fluorescence

Journal: Immunology

Article Title: NKG2A ‐checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA‐E

doi: 10.1111/imm.13515

Figure Lengend Snippet: NKG2A expression on CD8 + T cells and natural killer (NK) cells. (a) Left panel; representative gating of NKG2A and NKG2C on NK cells and CD8 + T cells from freshly isolated PBMCs is shown. Right panel; expression of NKG2A and NKG2C on CD8 + T cells and NK cells; each dot represents one donor. Median is shown ( n = 10). (b) Freshly isolated PBMCs were stimulated with K562S cells expressing CD86 for 5 days. NKG2A and NKG2C expression was assessed on NK and CD8 + T cells. Each dot represents one donor. Median is shown ( n = 6). (c) PBMCs were co‐cultured for 5 days with the indicated K562S cell lines and proliferation (CSFE low ) was analysed for NK cells. Left panel; one representative experiment is shown. Right panel; data from several donors are depicted (HLA‐G and HLA‐C4 [ n = 8]; HLA‐B8 and HLA‐A2 [ n = 4]). Each data point represents the mean of triplicates of one donor. NK cell proliferation induced upon stimulation with K562S expressing the indicated HLA‐E‐complexes is normalized to the NK cell proliferation induced by control K562S cells (% of CFSE low of K562S‐HLA‐E peptide stimulated NK cells/% of CFSE low control K562S‐stimulated NK cells). For statistical evaluation, a one‐way ANOVA followed by Dunn's multiple comparison test was performed (*** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; ns p > 0.05). (d) Lysis of the indicated target cells upon 4 h co‐culture with PBMCs pre‐stimulated for 7–10 days in the presence of IL‐2 and IL‐15 to induce NK cell expansion. Effector: target cell ratio was 10:1 and monalizumab was used at a final concentration of 5 μg/ml. Data were normalized to control K562 cells (control K562 = 100% cell lysis). Median is shown ( n = 10). For statistical evaluation, a two‐way ANOVA followed by Bonferroni post hoc test was performed (*** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; ns p > 0.05).

Article Snippet: Human NKG2A antibody (clone REA110) was from Miltenyi Biotec and clone Z199 was from Beckman Coulter Inc. Human NKG2A antibody (131411) and NKG2C antibody (134522) were from R&D systems.

Techniques: Expressing, Isolation, Cell Culture, Control, Comparison, Lysis, Co-Culture Assay, Concentration Assay

Journal: Immunology

Article Title: NKG2A ‐checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA‐E

doi: 10.1111/imm.13515

Figure Lengend Snippet: Evaluation of NKG2A antibodies in their binding and blocking capacities. (a) The EC 50 values were determined for the therapeutic NKG2A antibody monalizumab, as well as for the monoclonal antibodies clone Z199, clone REA110 and clone 131411. NKG2A expressing reporter cells were incubated with increasing concentrations ranging from 100 pg/ml to 10 μg/ml of the depicted antibodies. Antibody binding was detected with an APC‐conjugated anti‐human and anti‐mouse IgG antibody, respectively. EC 50 was calculated from six experiments performed in duplicate. (b) NKG2A expressing reporter cells were stimulated with control K562S and K562S‐HLA‐E HLA‐A2 in the presence or absence of monalizumab, Z199, REA110 and 131411 (all 1 μg/ml). Reporter activation is shown as fold induction (gMFI of K562S‐HLA‐E HLA‐A2 stimulated cells/gMFI control K562S‐stimulated cells). Results are shown of four (monalizumab) and two (Z199, REA110 and 131411) independent experiments performed in duplicates.

Article Snippet: Human NKG2A antibody (clone REA110) was from Miltenyi Biotec and clone Z199 was from Beckman Coulter Inc. Human NKG2A antibody (131411) and NKG2C antibody (134522) were from R&D systems.

Techniques: Binding Assay, Blocking Assay, Bioprocessing, Expressing, Incubation, Control, Activation Assay

Journal: Immunology

Article Title: NKG2A ‐checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA‐E

doi: 10.1111/imm.13515

Figure Lengend Snippet: Identification of the epitopes of NKG2A antibodies. (a) Sequence alignment of NKG2A (A113 to L233) and NKG2C (A111 to L231). (b) Schematic representations of wild‐type NKG2A and NKG2C, as well as chimeric constructs of NKG2A‐C (#1, #2, #3), are depicted. Cell surface expression of the molecules was determined by using a Strep‐tag antibody. (c) Jurkat cells expressing the indicated molecules were incubated with the following NKG2A antibodies monalizumab, REA110, Z199, 131411, and a NKG2C antibody (134522), respectively (open histogram: the indicated antibodies on control cells). (d) Schematic representation of a mutated NKG2C construct (NKG2C mut ) and its cell surface expression detected with a Strep‐tag antibody. (e) Flow cytometric analysis of the depicted antibodies on cells expressing the NKG2C mut molecule (open histogram: the indicated antibodies on control cells). (f) Crystal structure of NKG2A (orange) and CD94 (green) (PDB ID code 3BDW). Positions A113, M189 (light green), E197 (light green) and K232 are highlighted with arrows . One representative experiment is shown (b–e).

Article Snippet: Human NKG2A antibody (clone REA110) was from Miltenyi Biotec and clone Z199 was from Beckman Coulter Inc. Human NKG2A antibody (131411) and NKG2C antibody (134522) were from R&D systems.

Techniques: Sequencing, Construct, Expressing, Strep-tag, Incubation, Control

Journal: Immunology

Article Title: NKG2A ‐checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA‐E

doi: 10.1111/imm.13515

Figure Lengend Snippet: Assessment of functional EC 50 values of NKG2A blocking antibodies monalizumab and Z199. NKG2A/CD94 expressing reporter cells were stimulated with control K562S, K562S‐HLA‐E HLA‐G , K562S‐HLA‐E HLA‐B8 , K562S‐HLA‐E HLA‐A2 and K562S‐HLA‐E HLA‐C4 for 24 h in the presence of monalizumab (a) and Z199 (b) at concentrations ranging from 31.6 μg/ml to 10 ng/ml. White bars represent NKG2A inhibition in the absence of an antibody. Results are depicted of three (monalizumab) and two (Z199) independent experiments performed in duplicate. Left panels; data are normalized to control K562S cells in the absence or presence of the indicated antibody concentrations. Right panels; inhibition curves and half‐maximum effective concentrations (EC 50 ) were calculated for the NKG2A antibodies from normalized data.

Article Snippet: Human NKG2A antibody (clone REA110) was from Miltenyi Biotec and clone Z199 was from Beckman Coulter Inc. Human NKG2A antibody (131411) and NKG2C antibody (134522) were from R&D systems.

Techniques: Functional Assay, Blocking Assay, Expressing, Control, Inhibition

Journal: Immunology

Article Title: NKG2A ‐checkpoint inhibition and its blockade critically depends on peptides presented by its ligand HLA‐E

doi: 10.1111/imm.13515

Figure Lengend Snippet: EC 50 values and the 95% confidence intervals (CI) were determined for monalizumab and Z199 for their ability to block the HLA‐E peptide /NKG2A interaction in a functional assay.

Article Snippet: Human NKG2A antibody (clone REA110) was from Miltenyi Biotec and clone Z199 was from Beckman Coulter Inc. Human NKG2A antibody (131411) and NKG2C antibody (134522) were from R&D systems.

Techniques: Blocking Assay, Functional Assay

Journal:

Article Title: Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities

doi:

Figure Lengend Snippet: Akt phosphorylation is not inhibited by eIF4E overexpression. Tetracycline-inducible cells (S2-6, C1, C2, and C3) were incubated in the absence of tetracycline for the indicated times (lanes 1 through 12); NIH 3T3 cells were grown in Dulbecco’s minimal essential medium containing 0.5% fetal bovine serum (FBS) for 16 h (lanes 13 through 16), after which cells were either mock treated (lane 13), incubated for 30 min in the presence of 100 nM wortmannin (lane 15) or 20 ng of rapamycin/ml (lane 16) prior to stimulation with 10% FBS for 1 h, or stimulated with 10% FBS alone (lane 14). Cells were then harvested, and total cell extract (50 μg) was subjected to electrophoresis on an SDS–8% polyacrylamide gel, electroblotted, and probed by using the following: (A) goat polyclonal anti-Akt antibody (C-20), which recognizes Akt irrespective of its phosphorylation state; (B) mouse monoclonal anti-actin antibody; (C) rabbit polyclonal anti-phosphospecific (Ser473) Akt antibody. The result shown is a representative of two independent experiments.

Article Snippet: For each experiment, an equal amount of total protein (25 to 50 μg) was electrophoresed on a sodium dodecyl sulfate (SDS)–8 to 15% polyacrylamide gel and electroblotted onto nitrocellulose membranes (Millipore) in 25 mM Tris-HCl, pH 7.5; 190 mM glycine; and 20% (vol/vol) methanol, at 75 V for 2 h. Filters were then blocked in Tris-buffered saline containing 0.2% Tween 20 (TBST) and 5% (wt/vol) dry milk at 25°C for 1 h. Membranes were incubated overnight at 4°C with one of the following antibodies at the indicated dilution: mouse monoclonal antiactin at 1:500 (ICN Biomedicals), rabbit polyclonal anti-eIF4E at 1:1,000 ( 26 ), rabbit polyclonal anti-4E-BP1 at 1:1,000 ( 31 ), rabbit polyclonal anti-p70 S6k at 1:300 (Santa Cruz Biotechnology), rabbit polyclonal anti-phosphospecific p70 S6k (Thr421/Ser424) at 1:1,000 (New England Biolabs), rabbit polyclonal anti-phosphospecific Akt (Ser473) at 1:1,000 (New England Biolabs), and goat polyclonal anti-Akt (C-20) at 1:1,000 (Santa Cruz Biotechnology).

Techniques: Over Expression, Incubation, Electrophoresis

Journal:

Article Title: Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities

doi:

Figure Lengend Snippet: 4E-BP1 phosphorylation state in transformed NIH 3T3 cells. Immunoblot analysis of total cell extract (50 μg) obtained from parental NIH 3T3 cells or NIH 3T3 cells overexpressing eIF4E (eIF4E), eIF2αala mutant [2α (Ala)] and wild type [2α (wt)], and mutant PKRΔ6. The blot was probed with a rabbit polyclonal anti-4E-BP1 antibody, as described in Materials and Methods. The three isoforms of 4E-BP1 are indicated.

Article Snippet: For each experiment, an equal amount of total protein (25 to 50 μg) was electrophoresed on a sodium dodecyl sulfate (SDS)–8 to 15% polyacrylamide gel and electroblotted onto nitrocellulose membranes (Millipore) in 25 mM Tris-HCl, pH 7.5; 190 mM glycine; and 20% (vol/vol) methanol, at 75 V for 2 h. Filters were then blocked in Tris-buffered saline containing 0.2% Tween 20 (TBST) and 5% (wt/vol) dry milk at 25°C for 1 h. Membranes were incubated overnight at 4°C with one of the following antibodies at the indicated dilution: mouse monoclonal antiactin at 1:500 (ICN Biomedicals), rabbit polyclonal anti-eIF4E at 1:1,000 ( 26 ), rabbit polyclonal anti-4E-BP1 at 1:1,000 ( 31 ), rabbit polyclonal anti-p70 S6k at 1:300 (Santa Cruz Biotechnology), rabbit polyclonal anti-phosphospecific p70 S6k (Thr421/Ser424) at 1:1,000 (New England Biolabs), rabbit polyclonal anti-phosphospecific Akt (Ser473) at 1:1,000 (New England Biolabs), and goat polyclonal anti-Akt (C-20) at 1:1,000 (Santa Cruz Biotechnology).

Techniques: Transformation Assay, Western Blot, Mutagenesis

Journal:

Article Title: Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities

doi:

Figure Lengend Snippet: Expression of eIF4E in stably transfected tetracycline-inducible NIH 3T3 cells. (Upper panel) Immunoblot analysis was performed on total protein (25 μg) from cells cultured for 24 h in the presence or absence of tetracycline. S2-6 cells are the parental cells, which express tTA upon induction. C1, C2, and C3 are stably transfected cell lines expressing eIF4E. The blot was probed first with a rabbit polyclonal anti-eIF4E antibody, followed by a mouse monoclonal anti-actin antibody. After probing with 125I-labelled anti-rabbit and anti-mouse secondary antibodies, the amount of eIF4E expressed was quantitated with a Bas 2000 phosphorimager. Relative molecular masses for protein standards are indicated in kilodaltons to the right. (Lower panel) Quantitation of the results shown in the upper panel. eIF4E expression was normalized against actin levels and expressed as the ratio relative to the amount of eIF4E in S2-6 cells in the presence of tetracycline, which was set at 1. Values are the averages of three experiments, and the standard errors are shown.

Article Snippet: For each experiment, an equal amount of total protein (25 to 50 μg) was electrophoresed on a sodium dodecyl sulfate (SDS)–8 to 15% polyacrylamide gel and electroblotted onto nitrocellulose membranes (Millipore) in 25 mM Tris-HCl, pH 7.5; 190 mM glycine; and 20% (vol/vol) methanol, at 75 V for 2 h. Filters were then blocked in Tris-buffered saline containing 0.2% Tween 20 (TBST) and 5% (wt/vol) dry milk at 25°C for 1 h. Membranes were incubated overnight at 4°C with one of the following antibodies at the indicated dilution: mouse monoclonal antiactin at 1:500 (ICN Biomedicals), rabbit polyclonal anti-eIF4E at 1:1,000 ( 26 ), rabbit polyclonal anti-4E-BP1 at 1:1,000 ( 31 ), rabbit polyclonal anti-p70 S6k at 1:300 (Santa Cruz Biotechnology), rabbit polyclonal anti-phosphospecific p70 S6k (Thr421/Ser424) at 1:1,000 (New England Biolabs), rabbit polyclonal anti-phosphospecific Akt (Ser473) at 1:1,000 (New England Biolabs), and goat polyclonal anti-Akt (C-20) at 1:1,000 (Santa Cruz Biotechnology).

Techniques: Expressing, Stable Transfection, Transfection, Western Blot, Cell Culture, Quantitation Assay

Journal:

Article Title: Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities

doi:

Figure Lengend Snippet: Time course of induction of eIF4E. (Upper panel) Total cell extract (25 μg) was prepared from C1 cells cultured in the absence of tetracycline for the indicated times. Extracts were immunoblotted and probed with a rabbit polyclonal anti-eIF4E antibody. The blot was subsequently probed with a mouse monoclonal anti-actin antibody to normalize for protein loading. After probing with 125I-labelled anti-rabbit and anti-mouse secondary antibodies, the amount of eIF4E expressed was quantitated with a Bas 2000 phosphorimager. (Lower panel) Quantitative analysis of the results presented in the upper panel. eIF4E expression levels were normalized against actin levels and are expressed as the ratios relative to the amount of eIF4E in C1 cells at time 0, which was set at 1. Results are representative of two experiments for which the values differed by less than 14%.

Article Snippet: For each experiment, an equal amount of total protein (25 to 50 μg) was electrophoresed on a sodium dodecyl sulfate (SDS)–8 to 15% polyacrylamide gel and electroblotted onto nitrocellulose membranes (Millipore) in 25 mM Tris-HCl, pH 7.5; 190 mM glycine; and 20% (vol/vol) methanol, at 75 V for 2 h. Filters were then blocked in Tris-buffered saline containing 0.2% Tween 20 (TBST) and 5% (wt/vol) dry milk at 25°C for 1 h. Membranes were incubated overnight at 4°C with one of the following antibodies at the indicated dilution: mouse monoclonal antiactin at 1:500 (ICN Biomedicals), rabbit polyclonal anti-eIF4E at 1:1,000 ( 26 ), rabbit polyclonal anti-4E-BP1 at 1:1,000 ( 31 ), rabbit polyclonal anti-p70 S6k at 1:300 (Santa Cruz Biotechnology), rabbit polyclonal anti-phosphospecific p70 S6k (Thr421/Ser424) at 1:1,000 (New England Biolabs), rabbit polyclonal anti-phosphospecific Akt (Ser473) at 1:1,000 (New England Biolabs), and goat polyclonal anti-Akt (C-20) at 1:1,000 (Santa Cruz Biotechnology).

Techniques: Cell Culture, Expressing

Journal:

Article Title: Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities

doi:

Figure Lengend Snippet: eIF4E induction results in dephosphorylation of 4E-BP1. Cell extracts were prepared from C1 cells in the absence of tetracycline for the indicated times. (A) Total cell extract (25 μg) was resolved on an SDS–12.5% polyacrylamide gel, electroblotted onto a 0.45-μm-pore-size nitrocellulose membrane, and probed with rabbit polyclonal anti-eIF4E and mouse monoclonal anti-actin antibodies. (B) Total cell extract (50 μg) was resolved on an SDS–15% polyacrylamide gel, electroblotted onto a 0.2-μm-pore-size nitrocellulose membrane, and probed with a rabbit polyclonal anti-4E-BP1 antibody. (C) Far-Western analysis of cell extract (50 μg) by using 32P-labelled HMK-eIF4E as a probe was performed as described in Materials and Methods. In panels B and C, the different phosphorylation isoforms of 4E-BP1 are indicated with arrows.

Article Snippet: For each experiment, an equal amount of total protein (25 to 50 μg) was electrophoresed on a sodium dodecyl sulfate (SDS)–8 to 15% polyacrylamide gel and electroblotted onto nitrocellulose membranes (Millipore) in 25 mM Tris-HCl, pH 7.5; 190 mM glycine; and 20% (vol/vol) methanol, at 75 V for 2 h. Filters were then blocked in Tris-buffered saline containing 0.2% Tween 20 (TBST) and 5% (wt/vol) dry milk at 25°C for 1 h. Membranes were incubated overnight at 4°C with one of the following antibodies at the indicated dilution: mouse monoclonal antiactin at 1:500 (ICN Biomedicals), rabbit polyclonal anti-eIF4E at 1:1,000 ( 26 ), rabbit polyclonal anti-4E-BP1 at 1:1,000 ( 31 ), rabbit polyclonal anti-p70 S6k at 1:300 (Santa Cruz Biotechnology), rabbit polyclonal anti-phosphospecific p70 S6k (Thr421/Ser424) at 1:1,000 (New England Biolabs), rabbit polyclonal anti-phosphospecific Akt (Ser473) at 1:1,000 (New England Biolabs), and goat polyclonal anti-Akt (C-20) at 1:1,000 (Santa Cruz Biotechnology).

Techniques: De-Phosphorylation Assay, Western Blot

Journal:

Article Title: Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities

doi:

Figure Lengend Snippet: The extent of 4E-BP1 dephosphorylation is proportional to the level of eIF4E expression. Parental cells (S2-6) and eIF4E-expressing cell clones C1, C2, and C3 were cultured in the absence of tetracycline for the indicated times. Total cell extract (50 μg) was immunoblotted and probed with a rabbit polyclonal anti-4E-BP1 antibody. Arrows indicate the different phosphorylation isoforms of 4E-BP1.

Article Snippet: For each experiment, an equal amount of total protein (25 to 50 μg) was electrophoresed on a sodium dodecyl sulfate (SDS)–8 to 15% polyacrylamide gel and electroblotted onto nitrocellulose membranes (Millipore) in 25 mM Tris-HCl, pH 7.5; 190 mM glycine; and 20% (vol/vol) methanol, at 75 V for 2 h. Filters were then blocked in Tris-buffered saline containing 0.2% Tween 20 (TBST) and 5% (wt/vol) dry milk at 25°C for 1 h. Membranes were incubated overnight at 4°C with one of the following antibodies at the indicated dilution: mouse monoclonal antiactin at 1:500 (ICN Biomedicals), rabbit polyclonal anti-eIF4E at 1:1,000 ( 26 ), rabbit polyclonal anti-4E-BP1 at 1:1,000 ( 31 ), rabbit polyclonal anti-p70 S6k at 1:300 (Santa Cruz Biotechnology), rabbit polyclonal anti-phosphospecific p70 S6k (Thr421/Ser424) at 1:1,000 (New England Biolabs), rabbit polyclonal anti-phosphospecific Akt (Ser473) at 1:1,000 (New England Biolabs), and goat polyclonal anti-Akt (C-20) at 1:1,000 (Santa Cruz Biotechnology).

Techniques: De-Phosphorylation Assay, Expressing, Clone Assay, Cell Culture

Journal:

Article Title: Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities

doi:

Figure Lengend Snippet: Effect of eIF4E induction on p70S6k phosphorylation. eIF4E-expressing cells were maintained in the presence or absence of tetracycline for 36 h (lanes 1 through 6); NIH 3T3 cells were grown in Dulbecco’s minimal essential medium containing 0.5% fetal bovine serum (FBS) for 16 h (lanes 7 to 9), after which cells were either mock treated (lane 7), incubated for 30 min in the presence of 20 ng/ml of rapamycin prior to stimulation with 10% FBS for 1 h (lane 8), or stimulated with 10% FBS alone (lane 9). Total cell extract (50 μg) was electrophoresed on two separate SDS–8% polyacrylamide gels and electroblotted onto a 0.45-μm-pore-size nitrocellulose membrane. One membrane was probed with a rabbit polyclonal anti-p70S6k antibody which recognizes p70S6k irrespective of its phosphorylation state (A), while the other was probed with a rabbit polyclonal anti-phosphopeptide antibody which is specific for phospho-Thr421 and phospho-Ser424 in p70S6k (B). (C) Total cell extract (20 μg) was immunoprecipitated with rabbit polyclonal anti-p70S6k antibody. The immunoprecipitate was assayed for p70S6k activity by using 40S ribosomal subunits as a substrate as described in Materials and Methods. The figure is a representative of three independent experiments.

Article Snippet: For each experiment, an equal amount of total protein (25 to 50 μg) was electrophoresed on a sodium dodecyl sulfate (SDS)–8 to 15% polyacrylamide gel and electroblotted onto nitrocellulose membranes (Millipore) in 25 mM Tris-HCl, pH 7.5; 190 mM glycine; and 20% (vol/vol) methanol, at 75 V for 2 h. Filters were then blocked in Tris-buffered saline containing 0.2% Tween 20 (TBST) and 5% (wt/vol) dry milk at 25°C for 1 h. Membranes were incubated overnight at 4°C with one of the following antibodies at the indicated dilution: mouse monoclonal antiactin at 1:500 (ICN Biomedicals), rabbit polyclonal anti-eIF4E at 1:1,000 ( 26 ), rabbit polyclonal anti-4E-BP1 at 1:1,000 ( 31 ), rabbit polyclonal anti-p70 S6k at 1:300 (Santa Cruz Biotechnology), rabbit polyclonal anti-phosphospecific p70 S6k (Thr421/Ser424) at 1:1,000 (New England Biolabs), rabbit polyclonal anti-phosphospecific Akt (Ser473) at 1:1,000 (New England Biolabs), and goat polyclonal anti-Akt (C-20) at 1:1,000 (Santa Cruz Biotechnology).

Techniques: Expressing, Incubation, Immunoprecipitation, Activity Assay

Journal:

Article Title: Translational Homeostasis: Eukaryotic Translation Initiation Factor 4E Control of 4E-Binding Protein 1 and p70 S6 Kinase Activities

doi:

Figure Lengend Snippet: Proposed model for translational homeostasis in response to changes in eIF4E concentration. eIF4E incorporation into an eIF4F complex is regulated by the PI 3-kinase pathway, through Akt and FRAP/mTOR, which leads to the phosphorylation of 4E-BPs and p70S6k. Forced expression of eIF4E engenders a negative feedback loop by downregulation of a component of the Akt-FRAP/mTOR pathway or by activating a phosphatase (PPase) which dephosphorylates 4E-BP1 and p70S6k. This results in the dephosphorylation of p70S6k and 4E-BP1.

Article Snippet: For each experiment, an equal amount of total protein (25 to 50 μg) was electrophoresed on a sodium dodecyl sulfate (SDS)–8 to 15% polyacrylamide gel and electroblotted onto nitrocellulose membranes (Millipore) in 25 mM Tris-HCl, pH 7.5; 190 mM glycine; and 20% (vol/vol) methanol, at 75 V for 2 h. Filters were then blocked in Tris-buffered saline containing 0.2% Tween 20 (TBST) and 5% (wt/vol) dry milk at 25°C for 1 h. Membranes were incubated overnight at 4°C with one of the following antibodies at the indicated dilution: mouse monoclonal antiactin at 1:500 (ICN Biomedicals), rabbit polyclonal anti-eIF4E at 1:1,000 ( 26 ), rabbit polyclonal anti-4E-BP1 at 1:1,000 ( 31 ), rabbit polyclonal anti-p70 S6k at 1:300 (Santa Cruz Biotechnology), rabbit polyclonal anti-phosphospecific p70 S6k (Thr421/Ser424) at 1:1,000 (New England Biolabs), rabbit polyclonal anti-phosphospecific Akt (Ser473) at 1:1,000 (New England Biolabs), and goat polyclonal anti-Akt (C-20) at 1:1,000 (Santa Cruz Biotechnology).

Techniques: Concentration Assay, Expressing, De-Phosphorylation Assay

Journal: bioRxiv

Article Title: Haspin participates in Aurora phosphorylation at centromeres and contributes to chromosome congression in male mouse meiosis

doi: 10.1101/2021.11.02.466959

Figure Lengend Snippet: Double immunolabelling of H3T3ph (green) and SYCP3 (red) on squashed WT mouse spermatocytes on diplotene, (B) diakinesis, (C) prometaphase-I, (D) metaphase-I, (E) anaphase-I, (F) telophase-I, (G) interkinesis, (H) prometaphase-II, (I) metaphase-II, (J) anaphase-II and (K) telophase-II. Chromatin has been stained with DAPI (blue). Scale bar in K represents 10 µm.

Article Snippet: Histone modifications were detected with the following primary antibodies: rabbit polyclonal anti-H2AT120ph antibody (Active Motif, 39391) at a 1:10 dilution, rabbit polyclonal anti-H3T3ph antibodies (Abcam, ab-17532; and Upstate, 07-424) at a 1:800 dilution.

Techniques: Staining

Journal: bioRxiv

Article Title: Haspin participates in Aurora phosphorylation at centromeres and contributes to chromosome congression in male mouse meiosis

doi: 10.1101/2021.11.02.466959

Figure Lengend Snippet: a. Double immunolabelling of H3T3ph and SYCP3 in squashed spermatocytes for (A) control and (B) 2h, (C, D) 4h and (E) 8h 1 mM of LDN-192960. Scale bar in E represents 10 µm. b. Quantification of metaphase-I percentage with H3T3ph signal at different timings in cultured spermatocytes with 1 mM of LDN-192960. Data represents the percentage of metaphase-I with non-signal, dim or normal signal of H3T3ph for 2h, 4h and 8h of treatment. c. H3T3ph distribution in control and 1mM 6h LDN-192960-treated spermatocytes. Double immunolabelling of H3T3ph (green) and SYCP3 (red) in (A) control and (B-D) 1mM 6h LDN-192960-treated metaphase-I. Double immunolabelling of H3T3ph (green) and ACA (red) in (E) control and (F-H) 1mM 6h LDN-192960-treated metaphase-II. Chromatin has been stained with DAPI (blue). Scale bar in D and H represents 10 µm. d. Quantification of the incidence of misaligned metaphase-I and metaphase-II. Percentage of aligned, misaligned and apoptotic metaphases is represented for control and 1 mM LDN-192960 for 6h treatment. Experiments were conducted in three different biological replicates. Bars and error bars represent mean ± SD.

Article Snippet: Histone modifications were detected with the following primary antibodies: rabbit polyclonal anti-H2AT120ph antibody (Active Motif, 39391) at a 1:10 dilution, rabbit polyclonal anti-H3T3ph antibodies (Abcam, ab-17532; and Upstate, 07-424) at a 1:800 dilution.

Techniques: Control, Cell Culture, Staining

Journal: bioRxiv

Article Title: Haspin participates in Aurora phosphorylation at centromeres and contributes to chromosome congression in male mouse meiosis

doi: 10.1101/2021.11.02.466959

Figure Lengend Snippet: a. ICD components distribution during metaphase-I spermatocytes. Double immunolabelling of (A-B) H3T3ph (green) and SYCP3 (red), (C-D) AURKph (green) and SYCP3 (red), (E-F) H2AT120ph (green) and SYCP3 (red), and (G-H) SGO2 (green) and SYCP3 (red). Chromatin has been stained with DAPI (blue). Scale bar in H represents 10 µm. b. ICD components distribution during metaphase-II spermatocytes. Double immunolabelling of (A-B) H3T3ph (green) and SYCP3 (red), (C-D) AURKph (green) and SYCP3 (red), (E-F) H2AT120ph (green) and SYCP3 (red), and (G-H) SGO2 (green) and SYCP3 (red). Chromatin has been stained with DAPI (blue). Scale bar in H represents 10 µm. c. Quantitative analysis of ICD components signals in metaphase-I spermatocytes. Experiments were conducted for one biological replicates. Data represents mean ± SEM, **** p < 0.0001, Student’s t -test. d. Quantitative analysis of ICD components signals in metaphase-II spermatocytes. Experiments were conducted for one biological replicates. Data represents mean ± SEM, **** p < 0.0001, Student’s t -test.

Article Snippet: Histone modifications were detected with the following primary antibodies: rabbit polyclonal anti-H2AT120ph antibody (Active Motif, 39391) at a 1:10 dilution, rabbit polyclonal anti-H3T3ph antibodies (Abcam, ab-17532; and Upstate, 07-424) at a 1:800 dilution.

Techniques: Staining

Journal: bioRxiv

Article Title: Haspin participates in Aurora phosphorylation at centromeres and contributes to chromosome congression in male mouse meiosis

doi: 10.1101/2021.11.02.466959

Figure Lengend Snippet: a. Histological sections of seminiferous tubules and epididymis of Haspin +/+ and Haspin -/- . Scale bar represents 200 µm. b. Distribution of H3T3ph in Haspin +/+ and Haspin -/- spermatocytes. Double immunolabelling of H3T3ph (green) and SYCP3 (red) in (A) Haspin +/+ and (B-D) Haspin -/- metaphase-I. Double immunolabelling of H3T3ph (green) and SYCP3 (red) in (E) Haspin +/+ and (F-H) Haspin -/- metaphase-II. Chromatin has been stained with DAPI (blue). Scale bar in D and H represents 10 µm. c. Quantification of the incidence of misaligned metaphase-I and metaphase-II. Percentage of aligned, misaligned and apoptotic metaphase is represented for Haspin +/+ and Haspin -/- . Experiments were conducted in two different biological replicates. Bars and error bars represent mean ± SD.

Article Snippet: Histone modifications were detected with the following primary antibodies: rabbit polyclonal anti-H2AT120ph antibody (Active Motif, 39391) at a 1:10 dilution, rabbit polyclonal anti-H3T3ph antibodies (Abcam, ab-17532; and Upstate, 07-424) at a 1:800 dilution.

Techniques: Staining

Journal: bioRxiv

Article Title: Haspin participates in Aurora phosphorylation at centromeres and contributes to chromosome congression in male mouse meiosis

doi: 10.1101/2021.11.02.466959

Figure Lengend Snippet: a. ICD components distribution during metaphase-I. Double immunolabelling of (A-B) H3T3ph (green) and SYCP3 (red), (C-D) AURKph (green) and SYCP3 (red), (E-F) H2AT120ph (green) and SYCP3 (red), and (G-H) SGO2 (green) and SYCP3 (red). Chromatin has been stained with DAPI (blue). Scale bar in H represents 10 µm. b. ICD components distribution during metaphase-II spermatocytes. Double immunolabelling of (A-B) H3T3ph (green) and SYCP3 (red), (C-D) AURKph (green) and SYCP3 (red), (E-F) H2AT120ph (green) and SYCP3 (red), and (G-H) SGO2 (green) and SYCP3 (red). Chromatin has been stained with DAPI (blue). Scale bar in H represents 10 µm. c. Quantitative analysis of ICD components signals in metaphase-I spermatocytes. Experiments were conducted for two biological replicates. Data represents mean ± SEM, **** p < 0.0001, Student’s t -test. d. Quantitative analysis of ICD components signals in metaphase-II spermatocytes. Experiments were conducted for two biological replicates. Data represents mean ± SEM, **** p < 0.0001, Student’s t -test.

Article Snippet: Histone modifications were detected with the following primary antibodies: rabbit polyclonal anti-H2AT120ph antibody (Active Motif, 39391) at a 1:10 dilution, rabbit polyclonal anti-H3T3ph antibodies (Abcam, ab-17532; and Upstate, 07-424) at a 1:800 dilution.

Techniques: Staining

Journal: JCI Insight

Article Title: Complement factor B in high glucose–induced podocyte injury and diabetic kidney disease

doi: 10.1172/jci.insight.147716

Figure Lengend Snippet: ( A and B ) Representative immunohistochemical staining images showing the induction of p-STAT1 (Tyr701) and p-STAT1 (Ser727) in glomerulus from DKD human renal biopsies and mouse models. Scale bar: 20 μm. ( C and D ) Western blot assay showing the abundance of p-STAT1 (Tyr701) and p-STAT1 (Ser727) in mouse kidneys. ( E ) Representative immunohistochemical staining images showing the induction of p-STAT1 (Tyr701) and p-STAT1 (Ser727) in glomeruli among groups. Scale bar: 20 μm. ( F ) Western blot assay showing the abundance of p-S6, p-p70 S6K, p-4E-BP1, and p-mTOR in high glucose–cultured podocytes. ( G ) Western blot assay showing the abundance of p-STAT1 (Tyr701), p-STAT1 (Ser727), and CFB in high glucose–cultured podocytes treated with rapamycin (5 nM). ( H ) Representative immunofluorescence staining showing the abundance of p-S6, p-STAT1 (Ser727), and CFB in high glucose–cultured podocytes. Scale bar: 20 μm. ( I and J ) Western blot assay showing the abundance of CFB ( I ), p-S6, p-STAT1 (Tyr701), and p-STAT1 (Ser727) ( J ) in cultured podocytes treated with mTOR activator. ( K ) Western blot assay showing the abundance of p-STAT1 (Tyr701), p-STAT1 (Ser727), and CFB in cultured podocytes treated with fludarabine and mTOR activator (2 μM). ( L ) Western blot assay showing the abundance of p-STAT1 (Tyr701), p-STAT1 (Ser727), and CFB in high glucose–cultured podocytes treated with fludarabine (0.4 μM). ( M ) Quantitative analyses showing the abundance of CFB in high glucose–cultured podocytes treated with fludarabine (0.4 μM). # P < 0.05, * P < 0.05, n = 3. ( N ) Western blot assay showing the abundance of p-STAT1 (Tyr701), p-STAT1 (Ser727), and CFB in cultured podocytes treated with STAT1 siRNA and mTOR activator (2 μM). ( O ) Western blot assay showing the abundance of p-STAT1 (Tyr701), p-STAT1 (Ser727), and CFB in high glucose–cultured podocytes treated with STAT1 siRNA. Data are expressed as the mean ± SEM. Comparison between the groups was performed using 1-way ANOVA followed by the Tukey test.

Article Snippet: The primary antibodies were as follows: anti–p-S6 (catalog 4858, Cell Signaling Technology), anti-S6 (catalog 2217, Cell Signaling Technology), anti-Rheb1 (catalog ab25873, Abcam), anti–p-p70 S6K (Thr421/Ser424) (catalog 9204L, Cell Signaling Technology), anti-p70 S6K (catalog 9202, Cell Signaling Technology), anti–p-4E-BP (Thr37/46) (catalog 2855, Cell Signaling Technology), anti–4E-BP (catalog 9452, Cell Signaling Technology), anti–p-mTOR (catalog 5536, Cell Signaling Technology), anti-mTOR (catalog 2983, Cell Signaling Technology), anti-PP2Acα (catalog ab106262, Abcam), anti-CFB (catalog ab192577, Abcam), anti–p-STAT1 (Ser727) (catalog 8826S, Cell Signaling Technology) anti–p-STAT1 (Tyr701) (catalog 7649S, Cell Signaling Technology), anti-ubiquitin (catalog sc-8017, Santa Cruz Biotechnology), anti–β-actin (catalog sc-47778, Santa Cruz Biotechnology), and anti-GAPDH (catalog AP0063, Bioworld).

Techniques: Immunohistochemical staining, Staining, Western Blot, Cell Culture, Immunofluorescence, Comparison

Journal: JCI Insight

Article Title: Complement factor B in high glucose–induced podocyte injury and diabetic kidney disease

doi: 10.1172/jci.insight.147716

Figure Lengend Snippet: ( A ) Western blot assay showing the abundance of Rheb1 in cultured podocytes after high glucose treatment at different times. ( B ) GTP loading assay showing the induction of GTP-Rheb1 after high glucose treatment. ( C ) Western blot assay showing the abundance of p-S6, p-p70 S6K, p-4E-BP1, and p-mTOR in high glucose–cultured podocytes transfected with scramble or Rheb1 siRNA for 24 hours. ( D and E ) The cultured podocytes were pretransfected with scramble or Rheb1 siRNA for 24 hours, or treated with rapamycin for 30 minutes, followed by high glucose treatment for 24 hours. Propidium iodide (PI) staining ( D ) and quantitative analysis ( E ) of dead cells among different groups. Data are presented as the percentage of PI-staining-positive cells. # P < 0.05 vs. scramble control cells, n = 3; * P < 0.05, $ P < 0.05 vs. high glucose–treated podocytes, n = 3. Scale bar: 20 μm. ( F ) UACR in Podo-Rheb1 +/+ and Podo-Rheb1 –/– mice at 6 months after STZ-induced DM. * P < 0.05 vs. Podo-Rheb1 +/+ mice, n = 6–7. ( G ) Representative PAS staining and immunofluorescence staining for diabetic kidney injury, WT1, and nephrin among different groups. Scale bar: 50 μm. ( H – J ) Quantitative analyses of injury score, glomerular area, and WT1-positive podocytes per glomerulus among different groups. # P < 0.05 vs. vehicle control mice; * P < 0.05 vs. Podo-Rheb1 +/+ mice with STZ injection, n = 3–7. Data are expressed as the mean ± SEM. Comparison between the groups was performed using 1-way ANOVA followed by the Tukey test ( B and E ). Comparison between the groups was performed using the 2-tailed Student’s t test (paired t test) ( F ). Data in H – J were analyzed with 2-way ANOVA with Tukey’s post hoc test.

Article Snippet: The primary antibodies were as follows: anti–p-S6 (catalog 4858, Cell Signaling Technology), anti-S6 (catalog 2217, Cell Signaling Technology), anti-Rheb1 (catalog ab25873, Abcam), anti–p-p70 S6K (Thr421/Ser424) (catalog 9204L, Cell Signaling Technology), anti-p70 S6K (catalog 9202, Cell Signaling Technology), anti–p-4E-BP (Thr37/46) (catalog 2855, Cell Signaling Technology), anti–4E-BP (catalog 9452, Cell Signaling Technology), anti–p-mTOR (catalog 5536, Cell Signaling Technology), anti-mTOR (catalog 2983, Cell Signaling Technology), anti-PP2Acα (catalog ab106262, Abcam), anti-CFB (catalog ab192577, Abcam), anti–p-STAT1 (Ser727) (catalog 8826S, Cell Signaling Technology) anti–p-STAT1 (Tyr701) (catalog 7649S, Cell Signaling Technology), anti-ubiquitin (catalog sc-8017, Santa Cruz Biotechnology), anti–β-actin (catalog sc-47778, Santa Cruz Biotechnology), and anti-GAPDH (catalog AP0063, Bioworld).

Techniques: Western Blot, Cell Culture, Transfection, Staining, Control, Immunofluorescence, Injection, Comparison

Journal: JCI Insight

Article Title: Complement factor B in high glucose–induced podocyte injury and diabetic kidney disease

doi: 10.1172/jci.insight.147716

Figure Lengend Snippet: ( A ) Western blot assay showing the abundance of PP2Acα in cultured podocytes after high glucose treatment at different times. Relative change of PP2Acα/tubulin shown at the bottom of the bands. ( B ) Real-time PCR analysis showing the mRNA abundance for PP2Acα in cultured podocytes after high glucose treatment. * P < 0.05 and # P < 0.05 vs. control cells, n = 4. ( C ) Western blot assay showing the abundance of PP2Acα in cultured podocytes pretreated with lactacystin for 30 minutes, followed by high glucose treatment at different times. Relative change of PP2Acα/tubulin shown under the bands. ( D ) The cultured podocytes were pretreated with lactacystin for 30 minutes, followed by high glucose treatment for 15 minutes. Western blot assay showing the level of ubiquitin in the precipitates (PP2Acα-IP) and PP2Acα in lysates (input). ( E ) Western blotting assay showing the expression of PP2Acα (MR204384) in cultured podocytes after PP2Acα (MR204384) plasmid transfection. ( F and G ) Western blotting assays showing the abundance of p-S6, p-p70 S6K, p-4E-BP1, p-mTOR ( F ), p-Stat1 (Ser727), and CFB ( G ) in high glucose–cultured podocytes transfected with pcDNA3.0 or PP2Acα (MR204384) plasmid for 36 hours. ( H ) Western blotting analyses demonstrating the downregulation of PP2Acα after PP2Acα siRNA transfection. ( I and J ) Western blotting assays showing the abundance of p-S6, p-p70 S6K, p-4E-BP1, p-mTOR ( I ), p-Stat1 (Ser727), and CFB ( J ) in high glucose–cultured podocytes transfected with scramble or PP2Acα siRNA for 36 hours. Data are expressed as the mean ± SEM. Comparison between the groups was performed using 1-way ANOVA followed by the Tukey test.

Article Snippet: The primary antibodies were as follows: anti–p-S6 (catalog 4858, Cell Signaling Technology), anti-S6 (catalog 2217, Cell Signaling Technology), anti-Rheb1 (catalog ab25873, Abcam), anti–p-p70 S6K (Thr421/Ser424) (catalog 9204L, Cell Signaling Technology), anti-p70 S6K (catalog 9202, Cell Signaling Technology), anti–p-4E-BP (Thr37/46) (catalog 2855, Cell Signaling Technology), anti–4E-BP (catalog 9452, Cell Signaling Technology), anti–p-mTOR (catalog 5536, Cell Signaling Technology), anti-mTOR (catalog 2983, Cell Signaling Technology), anti-PP2Acα (catalog ab106262, Abcam), anti-CFB (catalog ab192577, Abcam), anti–p-STAT1 (Ser727) (catalog 8826S, Cell Signaling Technology) anti–p-STAT1 (Tyr701) (catalog 7649S, Cell Signaling Technology), anti-ubiquitin (catalog sc-8017, Santa Cruz Biotechnology), anti–β-actin (catalog sc-47778, Santa Cruz Biotechnology), and anti-GAPDH (catalog AP0063, Bioworld).

Techniques: Western Blot, Cell Culture, Real-time Polymerase Chain Reaction, Control, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Transfection, Comparison

Journal: Autophagy

Article Title: Harnessing autophagy for cell fate control gene therapy

doi: 10.4161/auto.24639

Figure Lengend Snippet: Figure 1. Autophagic phenotype of human T cells after treatment with rapamycin. CD4+ T cells were cultured in the presence of 1 μM rapamycin during 3 d and evaluated for autophagic markers. (A) The cells were lysed and subjected to western blotting using antibodies that detect both MAP1LC3B-I and MAP1LC3B-II (18 KDa and 16 KDa), as well as antibodies against SQSTM1 (62 KDa), phospho-RPS6KB1 (70 KDa) and ACTB (45 KDa). (B) Cells were also stained with MitoTracker Green FM (100 nM) and the mitochondrial mass examined by flow cytometry. A representative histogram is shown (left panel; Rapa treatment, black line; control, gray line; negative control, light gray line) and mean (± SEM) of the mean fluorescence intensity (MFI) of staining was determined by pooling results from three independent experiments (right panel; *p < 0.0374). (C) Mitochondrial membrane potential was evaluated by labeling cells with JC1 reagent and analyzing by flow cytometry at FL1 and FL2 channel. The ratio FL2 to FL1 (aggregate/monomer) was calculated and expressed as mean (± SEM) of four independent experiments (**p < 0.0014).

Article Snippet: Membranes were probed using anti-MAP1LC3B (Cell Signaling, 2775), anti-SQSTM1/p62 (Cell Signaling, 5114), anti-phospho RPS6KB1 (Thr421/Ser424; Cell Signaling, 9208) and anti-ACTB (Cell Signaling, 4967) antibodies, followed by anti-rabbit immunoglobulin.

Techniques: Cell Culture, Western Blot, Staining, Flow Cytometry, Control, Negative Control, Fluorescence, Membrane, Labeling

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 2. Inhibition of UVB-induced phosphorylation of Akt and p70 S6-K by pretreatment of cells with EGCG or theaflavins. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then pretreated with EGCG or theaflavins for 1 h at the indicated concentration. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. The cells were lysed, and the phosphorylation levels were estimated by immunoblotting.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Phospho-proteomics, Concentration Assay, Irradiation, Cell Culture, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 3. Inhibition of UVB-induced activation of Akt and p70 S6-K by pretreatment of cells with EGCG or theaflavins. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then pretreated with EGCG or theaflavins for 1 h at the indicated concentration. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. A, the cells were lysed, and Akt or p70 S6-K was immunopre- cipitated using anti-Akt1/PKB, PH domain antibody. The activity of Akt was assessed using Akt substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least three independent experiments. * and **, significant difference from the UVB control at p 0.05 and p 0.01, respectively. B, the cells were lysed, and p70 S6-K was immuno- precipitated using a p70 S6-K antibody. The activity of p70 S6-K was assessed using S6 substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least three independent experiments. *, signifi- cant difference from the UVB control at p 0.05.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Activation Assay, Concentration Assay, Irradiation, Cell Culture, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 4. Inhibition of UVB-induced phosphorylation of Akt and p70 S6-K by pretreatment of cells with U0126, SB202190, LY294002, or rapamycin. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then pretreated with U0126, SB202190, LY294002, or rapamycin for 1 h at the indicated concentration. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. The cells were lysed, and the phosphorylation levels were estimated by immunoblotting.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Phospho-proteomics, Concentration Assay, Irradiation, Cell Culture, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 5. Inhibition of UVB-induced activation of Akt and p70 S6-K by pretreatment of cells with U0126, SB202190, LY294002 or rapamycin. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then pretreated with U0126, SB202190, LY294002, or rapa- mycin for 1 h at the indicated concentration. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. A, the cells were lysed, and Akt or p70 S6-K was immunoprecipitated using a Akt1/PKB, PH domain antibody. The activity of Akt was assessed using Akt substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least two independent experiments. *, significant difference from the UVB control at p 0.01. B, the cells were lysed, and p70 S6-K was immunoprecipitated using a p70 S6-K antibody. The activity of p70 S6-K was assessed using S6 substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least three independent experiments. *, significant difference from the UVB con- trol at p 0.01.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Activation Assay, Concentration Assay, Irradiation, Cell Culture, Immunoprecipitation, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 6. UVB-induced phosphorylation and activation of p70 S6-K in cells expressing dominant negative mutant of JNK1. A, JB6 Cl 41 cells and its stable transfectants, Cl 41 CMV-neo, and Cl 41 dominant negative mutant of JNK1 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were then irradiated with UVB (4 kJ/m2) and cultured for 30 min. The cells were lysed, and the activity of JNKs was determined as described under “Experimental Procedures.” B, the transfectant cells were treated as described above and lysed. The levels of phosphoryla- tion of p70 S6-K were estimated by immunoblotting. C, JB6 Cl 41 and dominant negative mutant of JNK1 cells were treated as described above and lysed. p70 S6-K was immunoprecipitated from the lysates using a p70 S6-K antibody. The activities of p70 S6-K were assessed using S6 substrate peptide and [-32P]ATP. Each bar indicates the mean S.E. of at least two independent experiments.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Phospho-proteomics, Activation Assay, Expressing, Dominant Negative Mutation, Irradiation, Cell Culture, Activity Assay, Transfection, Western Blot, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 7. Direct inhibition of UVB-induced p70 S6-K activation by EGCG or theaflavins. JB6 Cl 41 cells (80% confluence) were starved by replacing the medium with 0.1% FBS MEM and culturing for 48 h. The cells were irradiated with UVB (4 kJ/m2) and subsequently cultured for 30 min. A, the cells were lysed, and Akt or p70 S6-K was immunoprecipitated using a Akt1/PKB, PH domain antibody. The activity of Akt was assessed using Akt substrate peptide and [-32P]ATP with different concentrations of EGCG or theaflavins. Each bar indicates the mean S.E. of at least three independent experi- ments. B, the cells were lysed, and p70 S6-K was immunoprecipitated using a p70 S6-K antibody. The activity of p70 S6-K was assessed using S6 substrate peptide and [-32P]ATP with different concentrations of EGCG or theaflavins. Each bar indicates the mean S.E. of at least three independent experiments. *, significant difference from the UVB control at p 0.05.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Inhibition, Activation Assay, Irradiation, Cell Culture, Immunoprecipitation, Activity Assay, Control

Journal: Journal of Biological Chemistry

Article Title: Inhibitory Mechanisms of Tea Polyphenols on the Ultraviolet B-activated Phosphatidylinositol 3-Kinase-dependent Pathway

doi: 10.1074/jbc.m107897200

Figure Lengend Snippet: FIG. 8. Effect of tea polyphenols on PI3K-dependent pathway induced by UVB irradiation. Exposure to UVB results in activation of the PI3K pathway. EGCG and theaflavins inhibit UVB-induced ac- tivation and phosphorylation of PI3K and its downstream effectors, Akt (Thr308/Ser473) and p70 S6-K (Thr389 and Thr421/Ser424). The tea poly- phenols did not affect phosphorylation of Akt (Thr308) by PDK-1. UVB- induced p70 S6-K activation is also blocked by EGCG or theaflavins. The Erk kinase pathway, critical for UVB-induced activities of Akt and p70 S6-K, is also inhibited by tea polyphenols. 1, activation; , inhi- bition; tea bags, tea polyphenols.

Article Snippet: Materials—Eagle’s minimal essential medium (MEM), fetal bovine serum (FBS), and gentamicin were from Whittaker Biosciences (Walkersville, MD); L-glutamine was from Life Technologies, Inc.; the PI3K inhibitor LY29402 was from Biomol (Plymouth Meeting, PA); the p70 S6-K inhibitor rapamycin, the p38 kinase inhibitor SB202190, and the MAP kinase/Erk kinase specific inhibitor U0126 were from Calbiochem (La Jolla, CA); the Akt immunoprecipitation kinase assay kit and S6 kinase assay kit were from Upstate Biotechnology Inc. (Lake Placid, NY); c-Jun fusion protein, Akt antibody, and phospho-specific Akt (Thr308 or Ser473) antibody, p70 S6-K antibody, and phospho-specific p70 S6-K (Thr389 and Thr421/Ser424) antibody, c-Jun (Ser63) antibody, and PhosphoPlus p44/42 MAP kinase, p38 kinase, and JNK kinase antibody kits were from Cell Signaling Technology Inc. (Beverly, MA); phospho-JNKs antibody (G-7) and agarose conjugated with monoclonal anti-phosphotyrosine antibody (PY20) were from Santa Cruz (Santa Cruz, CA); and phosphatidylinositol was from Sigma.

Techniques: Irradiation, Activation Assay, Phospho-proteomics