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: eLife

Article Title: DYRK1A interacts with the tuberous sclerosis complex and promotes mTORC1 activity

doi: 10.7554/eLife.88318

Figure Lengend Snippet:

Article Snippet: Antibody , anti- Phospho-p70 S6 Kinase (Thr389) (rabbit polyclonal) , Cell Signaling Technology , Cat# 9205 , WB (1:1000).

Techniques: Transfection, Construct, shRNA, Transduction, Knock-Out, Recombinant, Plasmid Preparation, Sequencing, Control, cDNA Synthesis, Software

Journal: Cell reports

Article Title: The FBXW7-SHOC2-Raptor Axis Controls the Cross-Talks between the RAS-ERK and mTORC1 Signaling Pathways

doi: 10.1016/j.celrep.2019.02.052

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ​ REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-SHOC2(T507) This paper N/A rabbit anti-S6Kα Santa Cruz Cat# sc-230; RRID:AB_632156 mouse anti-β-actin Santa Cruz Cat# sc-47778; RRID:AB_626632 rabbit anti-phospho-4EBP1 (Ser65) Cell Signaling Technology Cat# 9451; RRID:AB_330947 rabbit anti-phospho-4EBP1 (Thr37 / Thr46) Cell Signaling Technology Cat# 2855; RRID:AB_560835 rabbit anti-4EBP1 Cell Signaling Technology Cat# 9452;RRID:AB_331692 rabbit anti-phospho-S6K (Thr389) Cell Signaling Technology Cat# 9234; RRID:AB_2269803 rabbit anti-ATG5 Cell Signaling Technology Cat# 2630; RRID:AB_2062340 rabbit anti-p62 Cell Signaling Technology Cat# 5114; RRID:AB_10624872 rabbit anti-ERK1 and ERK2 Cell Signaling Technology Cat# 9107; RRID:AB_10695739 rabbit anti-phospho-ERK1 and ERK2 (T202/Y204) Cell Signaling Technology Cat# 4376; RRID:AB_331772 rabbit anti-phospho-ERK1 and ERK2 (T202/Y204) Cell Signaling Technology Cat# 4370; RRID:AB_2315112 mouse anti-MEK1 Cell Signaling Technology Cat# 2352; RRID:AB_10693788 rabbit anti-Raptor Cell Signaling Technology Cat# 2280; RRID:AB_561245 rabbit anti-Rictor Cell Signaling Technology Cat# 2114; RRID:AB_2179963 rabbit anti-mTOR Cell Signaling Technology Cat# 2983; RRID:AB_2105622 rabbit anti-LC3B Cell Signaling Technology Cat# 2775; RRID:AB_915950 rabbit anti-SHOC2 Abcam Cat# ab106430;AB_10861003 rabbit anti-FBXW7 Epitomics Cat# A301-721A; RRID:AB_1210898 goat anti-GST GE Lifescience Cat# 27457701V; RRID:AB_771432 rat anti-HA Roche Cat# 11867423001; RRID:AB_390918 mouse anti-Flag Sigma Cat# F1804; RRID:AB_262044 Alexa Fluor 488 Polyclonal Antibody Invitrogen Cat # A-11094; RRID:AB_221544 Biological Samples Human pancreatic cancer tissue microarrays (TMAs) containing 71 pairs of tumors and matched adjacent tissues and 28 tumors Shanghai Outdo Biotech.

Techniques: Recombinant, Luminescence Assay, Mutagenesis, Plasmid Preparation, Software