Journal: International Journal of Biological Sciences

Article Title: Cdh1 functions as an oncogene by inducing self-renewal of lung cancer stem-like cells via oncogenic pathways

doi: 10.7150/ijbs.38672

Figure Lengend Snippet: Primers for RT-qPCR

Article Snippet: The following primary antibodies were used: anti-E-cadherin (Proteintech, USA), anti-PI3K, anti-Akt, anti-mTOR, anti-S6K, anti-Eif4e, anti-Erk1/2, anti-p-Mek1/2, anti-p-Erk1/2, anti-p-Akt, anti-p-mTOR, anti-p-S6k (Cell Signaling Technology, USA), anti-Mek1 (Upstate, USA) and anti-GAPDH (Proteintech, USA).

Techniques:

Journal: Scientific Reports

Article Title: Alpha-ketoglutarate promotes skeletal muscle hypertrophy and protein synthesis through Akt/mTOR signaling pathways

doi: 10.1038/srep26802

Figure Lengend Snippet: C2C12 cells were cultured for 6 d in a differentiation medium. C2C12 myotubes were then exposed to different concentrations of AKG (0, 0.5, and 2 mM) for 48 h. ( a ) Total protein levels. ( b ) Puromycin levels detected in C2C12 myotubes by Western blot. ( c ) The phosphorylation levels of mTOR, S6, 4E-BP1, eIF4E, eIF2a, and MyHC by Western blot. ( d ) IHC analysis for MHCII and phospho-S6 in C2C12 myotubes. Data are presented as mean±S.E.M. Different superscripts “a”/“b”/“c” represent significant differences between groups ( P < 0.05), and * means P < 0.05 compared with the control. β-actin served as a housekeeping gene.

Article Snippet: The PVDF membranes were then incubated with the indicated antibodies, including rabbit anti-β-actin (Bioss) and mouse puromycin antibody 12D10 (Millipore); or rabbit anti-phospho-mTOR (Ser2481) and mTOR, rabbit anti-phosphor-P70S6K (Thr389) and P70S6K1, rabbit anti-phospho-S6 (Ser235/236) and S6, rabbit anti-phospho-4E-BP1 (Thr37/46) and 4E-BP1, rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308;), rabbit anti-eIF4E, rabbit anti-phospho-eIF4E (Ser209), rabbit anti-eIF2α, rabbit anti-phospho-eIF2α (Ser251), rabbit anti-FoxO1, and rabbit anti-phospho-FoxO1 (Ser256) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Cell Culture, Western Blot, Phospho-proteomics, Control

Journal: Scientific Reports

Article Title: Alpha-ketoglutarate promotes skeletal muscle hypertrophy and protein synthesis through Akt/mTOR signaling pathways

doi: 10.1038/srep26802

Figure Lengend Snippet: ( a ) C2C12 myotubes were treated with 2 mM AKG for 1, 2, and 4 h. The expression levels of phosphorylation of mTOR, S6K, S6, 4E-BP1, eIF2a, and eIF4E were detected by Western blot. ( b ) C2C12 cells were cultured for 6 d in a differentiation medium. mTOR inhibitor rapamycin (1 μM) was used alone or co-treated with AKG (2 mM) for 48 h. Total protein levels. ( c ) The expression of puromycin determined by Western blot after C2C12 cells were co-treated with AKG and rapamycin. ( d ) The expression levels of phosphorylation of mTOR, S6, and 4E-BP1 detected by Western blot after C2C12 cells were co-treated with AKG and rapamycin. Data are presented as mean ± SEM. *means P < 0.05 compared with the control. β-actin served as a housekeeping gene control.

Article Snippet: The PVDF membranes were then incubated with the indicated antibodies, including rabbit anti-β-actin (Bioss) and mouse puromycin antibody 12D10 (Millipore); or rabbit anti-phospho-mTOR (Ser2481) and mTOR, rabbit anti-phosphor-P70S6K (Thr389) and P70S6K1, rabbit anti-phospho-S6 (Ser235/236) and S6, rabbit anti-phospho-4E-BP1 (Thr37/46) and 4E-BP1, rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308;), rabbit anti-eIF4E, rabbit anti-phospho-eIF4E (Ser209), rabbit anti-eIF2α, rabbit anti-phospho-eIF2α (Ser251), rabbit anti-FoxO1, and rabbit anti-phospho-FoxO1 (Ser256) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Expressing, Phospho-proteomics, Western Blot, Cell Culture, Control

Journal: Scientific Reports

Article Title: Alpha-ketoglutarate promotes skeletal muscle hypertrophy and protein synthesis through Akt/mTOR signaling pathways

doi: 10.1038/srep26802

Figure Lengend Snippet: AKG (0.6 g/kg) and puromycin were co-injected for 3 h. Protein turnover associate protein expression was detected by Western blot. ( a ) The expression of puramycin was analyzed by Western blot. ( b ) The expression of MyHC and the phosphorylation levels of mTOR, S6, 4E-BP1, eIF4E, eIF2a in the gastrocnemius of mice. ( c ) The expression of Akt, MAFbx, and MuRF1. ( d ) The mRNA expression levels of protein turnover related genes were measured by qPCR. ( e ) The phosphorylation levels of Akt and mTOR 1 h after AKG injection detected by Western blot. Data are presented as mean ± SEM. *means P < 0.05 compared with the control. β-actin served as a housekeeping gene control.

Article Snippet: The PVDF membranes were then incubated with the indicated antibodies, including rabbit anti-β-actin (Bioss) and mouse puromycin antibody 12D10 (Millipore); or rabbit anti-phospho-mTOR (Ser2481) and mTOR, rabbit anti-phosphor-P70S6K (Thr389) and P70S6K1, rabbit anti-phospho-S6 (Ser235/236) and S6, rabbit anti-phospho-4E-BP1 (Thr37/46) and 4E-BP1, rabbit anti-Akt, rabbit anti-phospho-Akt (Ser473), rabbit anti-phospho-Akt (Thr308;), rabbit anti-eIF4E, rabbit anti-phospho-eIF4E (Ser209), rabbit anti-eIF2α, rabbit anti-phospho-eIF2α (Ser251), rabbit anti-FoxO1, and rabbit anti-phospho-FoxO1 (Ser256) (Cell Signaling Technology, Beverly, MA, USA).

Techniques: Injection, Expressing, Western Blot, Phospho-proteomics, Control

Journal: Cell Death & Disease

Article Title: Glycerol-3-phosphate acyltransferase-1 upregulation by O-GlcNAcylation of Sp1 protects against hypoxia-induced mouse embryonic stem cell apoptosis via mTOR activation

doi: 10.1038/cddis.2015.410

Figure Lengend Snippet: Involvement of mTOR in GPAT1-induced mESCs anti-apoptosis under hypoxia. ( a ) Cells were transfected with gpat1 and NT siRNA for 24 h before glucosamine (10 μ M) for 30 min. Subsequently, cells were exposed to hypoxia treatment for 24 h. Collected samples are lysed, and p-mTOR, GPAT1, and β -actin protein expressions were measured by using western blotting. Each result shown is representative of three independent experiments. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with glucosamine. ( b ) Cells were pretreated with rapamycin (10 nM) before glucosamine (10 μ M) treatment; and then, cells were exposed to hypoxia for 24 h. Total proteins were extracted, and blotted with Bcl-2, Bax, cleaved caspase-9, and β -actin. Each result shown is representative of three independent experiments. ( c ) Cells were pretreated with glucosamine and/or rapamycin (10 nM) for 30 min before hypoxia treatment, and cytochrome c, COX IV, and β -tubulin in the cytosolic and mitochondrial fraction were detected by western blot. ( d ) Cells were pretreated with various concentrations of LPA (10 − 6 M–10 − 9 M) before hypoxia treatment for 24 h. Cell viability was measured by trypan blue exclusion assay. Error bars are presented as a mean±S.E.M. of three independent duplex dishes. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone. ( e ) Cells were pretreated with pertussis toxin (100 ng/ml) for 30 min before LPA treatment (0.1 μ M) for 30 min. Subsequently, cells were exposed to hypoxia treatment for 24 h. Total proteins were extracted and blotted with p-mTOR, mTOR, p-S6K1, S6K1, p-4EBP1, 4EBP1, and β -actin. Each result shown is representative of three independent experiments. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. ( f ) p-mTOR was immunostained with p-mTOR antibody, and counter-stained with PI. Fluorescence images were acquired by using confocal microscopy. Fluorescence intensity of p-mTOR was quantified by using ImageJ software. Data are presented as a mean±S.E.M. of three independent experiments. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone. ( g ) Cells were pretreated with rapamycin (10 nM) before LPA (0.1 μ M) treatment; and then, cells were exposed to hypoxia for 24 h. Total proteins were extracted, and blotted with Bcl-2, Bax, cleaved caspase-9, cleaved caspase-3, and β -actin. Each result shown is representative of three independent experiments. ( h ) Cell viability was measured by using cell counter. Data are presented as a mean±S.E.M. of three independent duplex dishes. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. ( i ) Viable cells were measured by using annexin V/PI flow cytometry analysis. Annexin V-negative-PI-negative cells (Q3) were considered viable, annexin V-negative-PI-positive cells (Q1) were considered necrotic, annexin V-positive-PI-positive cells (Q2) were considered late apoptotic, and annexin V-positive-PI-negative cells (Q4) were considered early apoptotic. Data are presented as a mean±S.E.M. of two independent duplex dishes. * P <0.05 versus control, # P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. * P <0.05 versus control, # indicates P <0.05 versus hypoxia treatment alone, and @ P <0.05 versus hypoxia with LPA. The proposed model for signaling pathways involved in glucosamine-induced mESCs survival under hypoxia ( j )

Article Snippet: Mammalian target of rapamycin (mTOR), p-mTOR (Ser 2448), S6K1, p-S6K1 (Thr 389), 4EBP1, and p-4EBP1 (Thr 37/46) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Western Blot, Control, Trypan Blue Exclusion Assay, Staining, Fluorescence, Confocal Microscopy, Software, Flow Cytometry, Protein-Protein interactions

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Mammalian Target of Rapamycin Induces Phosphatidylinositol 3-Kinase-Dependent and Mnk-Mediated Eukaryotic Translation Initiation Factor 4E Phosphorylation

doi: 10.1128/mcb.00760-07

Figure Lengend Snippet: FIG. 1. Rapamycin increases eIF4E phosphorylation under serum-free conditions (A), in the presence of IGF-1 (B) and an IGF-1R inhibitor (C), and in IRS-1-silenced (D) or -deficient (E) cells. (A) H157 cells were serum starved for 24 h and then treated with 10 nM rapamycin in the absence of FBS for the indicated times. In addition, H157 cells were cultured and treated with 10 nM rapamycin in medium containing 5% FBS for the given times. (B) H157 cells were serum starved for 20 h and then treated with 10 nM rapamycin alone, 1 ng/ml of IGF-1 alone, and their combination for 30, 60, 120, and 180 min. (C) A549 cells were pretreated with the given concentrations of IGF-1R inhibitor II (IGF-1Ri-II) for 30 min and then cotreated with 10 nM rapamycin (Rap) for the indicated times. (D) A549 cells were transfected with control (Ctrl) or IRS-1 siRNA. After 48 h, the cells were treated with 10 nM rapamycin (Rap) for 1 h before they were harvested for preparation of whole-cell protein lysates. In addition, untransfected cells (No) were included as a control. (E) WT and IRS/ murine 3T3 fibroblasts were treated with 10 nM rapamycin (R) or RAD001 (R1) for 8 h. After the aforementioned treatments, the cells were subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis.

Article Snippet: Rabbit polyclonal antibodies against phospho-Akt (p-Akt) (Ser473), phospho-p70S6K (p-p70S6K) (Thr389), phospho-Mnk1 (Thr197/202), eIF4E, and phospho-eIF4E (p-eIF4E) (Ser209), respectively, were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Techniques: Phospho-proteomics, Cell Culture, Transfection, Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Mammalian Target of Rapamycin Induces Phosphatidylinositol 3-Kinase-Dependent and Mnk-Mediated Eukaryotic Translation Initiation Factor 4E Phosphorylation

doi: 10.1128/mcb.00760-07

Figure Lengend Snippet: FIG. 2. Effects of knockdown of mTOR (A, B, and D), raptor (C and D), rictor (D), or p70S6K (E) on basal levels of eIF4E phosphorylation and rapamycin-induced eIF4E phosphorylation. (A) H157 cells were transfected with control or mTOR siRNA. After 48 h, the cells were subjected to preparation of whole-cell protein lysates. (B) H157 cells were transfected with control or mTOR siRNA for 48 h. Before the cells were harvested for preparation of whole-cell protein lysates, the cells were treated with 10 nM rapamycin for 3 h. (C) H157 cells were transfected with control or raptor siRNA. After the indicated times, the cells were subjected to preparation of whole-cell protein lysates. (D) Calu-1 cells were infected once with lentivirus carrying control, mTOR, raptor, or rictor shRNA and then subjected to selection with 1 g/ml puromycin for 10 days. The surviving cells were further cultured in puromycin-free medium. After another 10 days, the cells were seeded, treated with 10 nM rapamycin for 1 h, and then harvested for preparation of whole-cell protein lysates and subsequent Western blot analysis. (E) H157 cells were transfected with control or p70S6K siRNA for the indicated times. Before the cells were harvested at each time point for preparation of whole-cell protein lysates, the cells were treated with 10 nM rapamycin for 3 h. The indicated proteins in these experiments were detected by Western blot analysis. LE, longer exposure; Ctrl, control; TOR, mTOR; Rap, rapamycin.

Article Snippet: Rabbit polyclonal antibodies against phospho-Akt (p-Akt) (Ser473), phospho-p70S6K (p-p70S6K) (Thr389), phospho-Mnk1 (Thr197/202), eIF4E, and phospho-eIF4E (p-eIF4E) (Ser209), respectively, were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Techniques: Knockdown, Phospho-proteomics, Transfection, Control, Infection, shRNA, Selection, Cell Culture, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Mammalian Target of Rapamycin Induces Phosphatidylinositol 3-Kinase-Dependent and Mnk-Mediated Eukaryotic Translation Initiation Factor 4E Phosphorylation

doi: 10.1128/mcb.00760-07

Figure Lengend Snippet: FIG. 3. Effects of Akt knockdown on eIF4E phosphorylation (A) or rapamycin-induced eIF4E phosphorylation (B). (A) H157 cells were transfected with control (Ctrl) or Akt siRNA. After 48 h, the cells were subjected to preparation of whole-cell protein lysates. (B) H157 cells were transfected with control (Ctrl) or Akt siRNA for the indi- cated times. Before the cells were harvested for preparation of whole- cell protein lysates, the cells were treated with 10 nM rapamycin (Rap) for 3 h. The indicated proteins in these experiments were detected by Western blot analysis.

Article Snippet: Rabbit polyclonal antibodies against phospho-Akt (p-Akt) (Ser473), phospho-p70S6K (p-p70S6K) (Thr389), phospho-Mnk1 (Thr197/202), eIF4E, and phospho-eIF4E (p-eIF4E) (Ser209), respectively, were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Techniques: Knockdown, Phospho-proteomics, Transfection, Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Mammalian Target of Rapamycin Induces Phosphatidylinositol 3-Kinase-Dependent and Mnk-Mediated Eukaryotic Translation Initiation Factor 4E Phosphorylation

doi: 10.1128/mcb.00760-07

Figure Lengend Snippet: FIG. 4. mTOR inhibitors induce PI3K-dependent eIF4E phosphorylation. (A and B) Modulation of eIF4E phosphorylation by RAD001 in the presence of the PI3K inhibitor LY294002 (A) or wortmannin (Wort.) (B). H157 (A) or H157 and A549 (B) cells were treated with 1 nM RAD001 in the absence and presence of the indicated concentrations of LY194002 (A) or wortmannin (B) for 3 h. (C) Impact of p85 deficiency on rapamycin-induced eIF4E phosphorylation. The WT and p85-DKO MEFs were treated with 10 nM rapamycin for the indicated times. (D) Effects of expressing a constitutively active p110 catalytic subunit of PI3K on eIF4E phosphorylation. The indicated cancer cell lines were plated in six-well cell culture plates and then infected with Ad-GFP or Ad-p110* for 24 h. (E) Effects of rapamycin on PI3K activity. The indicated cancer cell lines were treated with 10 nM rapamycin (Rap) for 1 h. After the aforementioned treatments, the cell lines were subjected to preparation of whole-cell protein lysates and subsequent Western blot analysis for detection of the proteins as presented (A to D) or PI3K activity assay (E). L.E., longer exposure; EGF, epidermal growth factor.

Article Snippet: Rabbit polyclonal antibodies against phospho-Akt (p-Akt) (Ser473), phospho-p70S6K (p-p70S6K) (Thr389), phospho-Mnk1 (Thr197/202), eIF4E, and phospho-eIF4E (p-eIF4E) (Ser209), respectively, were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Techniques: Phospho-proteomics, Expressing, Cell Culture, Infection, Activity Assay, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Mammalian Target of Rapamycin Induces Phosphatidylinositol 3-Kinase-Dependent and Mnk-Mediated Eukaryotic Translation Initiation Factor 4E Phosphorylation

doi: 10.1128/mcb.00760-07

Figure Lengend Snippet: FIG. 5. mTOR inhibitors increase eIF4E phosphorylation through a Mnk-dependent mechanism. (A) Knockdown of Mnk1 does not abrogate rapamycin-induced eIF4E phosphorylation. H157 cells that were not transfected (NT) or were transfected with control (Ctrl) or Mnk1 siRNA for 48 h were treated with 10 nM rapamycin (Rap). After 3 h, the cells were subjected to preparation of whole-cell protein lysates. (B) Modulation of eIF4E phosphorylation by mTOR inhibitors in WT, 1-KO, 2-KO, and Mnk1/Mnk2 DKO cells. The indicated MEF lines were treated with 10 nM rapamycin (R) or RAD001 (R1) for 3 h and then subjected to preparation of whole-cell protein lysates. The indicated proteins in these experiments were detected by Western blot analysis.

Article Snippet: Rabbit polyclonal antibodies against phospho-Akt (p-Akt) (Ser473), phospho-p70S6K (p-p70S6K) (Thr389), phospho-Mnk1 (Thr197/202), eIF4E, and phospho-eIF4E (p-eIF4E) (Ser209), respectively, were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Techniques: Phospho-proteomics, Knockdown, Transfection, Control, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Mammalian Target of Rapamycin Induces Phosphatidylinositol 3-Kinase-Dependent and Mnk-Mediated Eukaryotic Translation Initiation Factor 4E Phosphorylation

doi: 10.1128/mcb.00760-07

Figure Lengend Snippet: FIG. 6. Inhibition of Mnk-dependent eIF4E phosphorylation by a Mnk1 inhibitor (C) augments rapamycin-mediated growth inhibition (A and B) in human lung cancer cells. (A) The individual cell lines, as indicated, were seeded in 96-well plates. On the next day, they were treated with the indicated concentrations of rapamycin (Rap) alone, 2.5 M CGP57380 (CGP) alone, and their combination. After 3 days, the plates were subjected to determination of cell numbers using the sulforhodamine B assay. The bars are means of four replicate determinations plus standard deviations. (B) H460 cells at a density of 250 cells per well were seeded in 12-well plates. On the second day, the cells were treated with the indicated concentrations of rapamycin (Rap) alone, CGP57380 (CGP) alone, and their combination. The same treatments were repeated every 3 days. After 10 days, the plates were stained for the formation of cell colonies with crystal violet. The colonies in each well were counted. The bars are means of three replicate determinations plus standard deviations. (C) H157 cells were pretreated with the indicated concentrations of CGP57380 (CGP) for 30 min and then cotreated with 10 nM rapamycin (Rap) for 1 h or 24 h. The cells were then subjected to preparation of whole-cell protein lysates for detection of the indicated proteins using Western blotting.

Article Snippet: Rabbit polyclonal antibodies against phospho-Akt (p-Akt) (Ser473), phospho-p70S6K (p-p70S6K) (Thr389), phospho-Mnk1 (Thr197/202), eIF4E, and phospho-eIF4E (p-eIF4E) (Ser209), respectively, were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Techniques: Inhibition, Phospho-proteomics, Sulforhodamine B Assay, Staining, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Mammalian Target of Rapamycin Induces Phosphatidylinositol 3-Kinase-Dependent and Mnk-Mediated Eukaryotic Translation Initiation Factor 4E Phosphorylation

doi: 10.1128/mcb.00760-07

Figure Lengend Snippet: FIG. 7. Schema for PI3K-dependent and Mnk-mediated eIF4E phosphorylation during mTOR inhibition in human cancer cells. In the literature, it has been suggested that mTOR/raptor exerts feedback inhibition of PI3K/Akt via p70S6K-mediated phosphorylation and deg- radation of IRS-1. Our results in this study suggest that mTOR/raptor can also negatively regulate PI3K activity through an unknown mech- anism independently of p70S6K/IRS-1. Moreover, we suggest that PI3K may regulate Mnk activity. Thus, we propose that inhibition of mTOR/raptor with an mTOR inhibitor (e.g., rapamycin) can increase PI3K activity independently of p70S6K/IRS-1, resulting in a Mnk- mediated increase in eIF4E phosphorylation.

Article Snippet: Rabbit polyclonal antibodies against phospho-Akt (p-Akt) (Ser473), phospho-p70S6K (p-p70S6K) (Thr389), phospho-Mnk1 (Thr197/202), eIF4E, and phospho-eIF4E (p-eIF4E) (Ser209), respectively, were purchased from Cell Signaling Technology, Inc. (Beverly, MA).

Techniques: Phospho-proteomics, Inhibition, Activity Assay

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 1. CBP80 and eIF4E are bound to mRNA in both nuclear and cytoplasmic cell fractions. (A) Structure of the pmCMV-Gl test plasmids, which harbor either a nonsense-free Gl allele that terminates translation at codon 147 (Norm) or a Gl allele carrying a TAG nonsense codon at position 39 (Ter). (B) Cos cells were transiently transfected with a test pmCMV-Gl plasmid and the reference plasmid phCMV-MUP. Nuclear (N) and cytoplasmic (C) fractions were then immunopurified under conditions that preserved RNP using either normal rabbit serum (NRS), anti-CBP80 antibody (α-CBP80) or anti-eIF4E antibody (α-eIF4E), and subjected to western blotting using anti-CBP80 antibody or anti-eIF4E antibody. Nuclear fractions were free of cytoplasm as evidenced by the absence of detectable reactivity with anti-eIF4A antibody (data not shown). For easy visualization, bands corresponding to antibody are not shown. (C) The levels of Gl and MUP mRNAs were quantitated in RNA prepared from each immunopurification using RT–PCR. The leftmost five lanes, which analyze decreasing amounts of RNA before immunopurification (– IP), demonstrate that the conditions of RT–PCR were quantitative. Numbers below the figure represent the level of Gl mRNA normalized to the level of MUP mRNA, where normalized levels of nonsense-free (Norm) mRNA in each anti-CBP80 and anti-eIF4E immunopurification of each fraction are defined as 100%. Results are representative of two independently performed experiments where the efficiency of NMD varied by no more than 12%. The immunopurification using anti-eIF4E and cytoplasmic sample was less efficient for the transfection using pmCMV-Gl Norm than for the transfection using pmCMV-Gl Ter, explaining the reduced levels of eIF4E detected by western blotting (B) and RNA detected by RT–PCR (C).

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Transfection, Plasmid Preparation, Western Blot, Immu-Puri, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 2. CBP80 but not eIF4E is detectably bound to intron-containing pre-mRNA. (A) As in Figure 1, except that only nuclear fractions and Gl pre-mRNA rather than Gl mRNA were analyzed. Furthermore, mouse anti-eIF4E antibody was used instead of rabbit anti-eIF4E antibody and mouse IgG served to control for non-specific immunopurification using mouse anti-eIF4E antibody. (B) As in (A) except that rabbit anti-eIF4E antibody was used in place of mouse anti-eIF4E antibody and the analysis of MUP mRNA was omitted. (C) As in (A) except that SV40 pre-mRNA and mRNA were analyzed. Results are representative of other independently performed experiments, including those using rabbit anti-eIF4E antibody and NRS as a control. Taking the sum of Gl or SV40 pre-mRNA immunopurified by anti-CBP80 antibody and anti-eIF4E antibody in each panel as 100%, the amount of CBP80-bound pre-mRNA was 96 ± 6% and the amount of eIF4E-bound pre-mRNA was 4 ± 6%.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Immu-Puri

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 3. CBP80 but neither eIF4E nor Upf2 immunopurifies with the CTD. The nuclear fraction of 32 × 106 untransfected Cos cells was immunopurified using either NRS or antibody to the C-terminal domain of RNA polymerase II (α-CTD), and protein from either 8 × 105 cells (CTD) or 32 × 105 cells (CBP80, eIF4E and Upf2) was subjected to western blotting using anti-CTD, anti-CBP80, anti-eIF4E or anti-Upf2 antibody. The three leftmost lanes, which analyze 2-fold dilutions of nuclear protein from 1.6 × 105 cells (CTD and CBP80) or 6.4 × 105 cells (eIF4E and Upf2) before immunopurification (– IP), demonstrate that the conditions of western blotting were semi-quantitative. The two forms of CTD probably differ in the degree of phosphorylation (Dubois et al., 1994). Results are representative of 2–4 independently performed experiments, depending on the antibody used in western blotting.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Western Blot, Immu-Puri

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 5. RNPS1, Y14, SRm160, REF/Aly and Upf2 co-immunopurify with Upf3X in an RNase-insensitive manner, while RNPS1, Y14, REF/Aly, TAP, Upf3X and Upf2 co-immunopurify with CBP80 in an RNase-sensitive manner. As in Figure 4 except that an additional immunopurification using anti-Upf3/3X was performed, total (T) lysate was immunopurified using anti-CBP80 antibody, RNase was added to half of each sample prior to immunopurification, RNA as well as protein were prepared, and western blot analyses of eIF4E and DEK were omitted. (A) RT–PCR quantitation of the level of β-actin mRNA before or after immunopurification using anti-Upf3/3X antibody in order to demonstrate the efficiency of RNase treatment. Prior to immununopurification, half of the sample was treated with RNase (+), and the other half was not treated with RNase (–). (B) Western blot analysis of protein immunopurified using anti-Upf3/3X antibody from nuclear and cytoplasmic fractions. (C) Western blot analysis of protein immunopurified using anti-CBP80 antibody from total cell extract.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Immu-Puri, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitation Assay

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 4. Nuclear and cytoplasmic CBP80, unlike nuclear and cytoplasmic eIF4E, co-immunopurify with RNPS1, Y14, SRm160, REF/Aly, TAP, Upf3X and Upf2. Nuclear and cytoplasmic fractions of 32 × 106 untransfected Cos cells were immunopurified using anti-CBP80 antibody, anti-eIF4E antibody or, as a control, NRS. Immunopurified protein from 8 × 105 or 32 × 105 cells was then analyzed by western blotting using the antibodies specified. The three leftmost lanes, which analyze 2-fold dilutions of nuclear protein from 8 × 105 or 32 × 105 cells before immunopurification (– IP), demonstrate that the conditions of western blotting were semi-quantitative. Notably, Upf3 probably co-migrates with the heavy chain of anti-hUpf3/3X antibody and, therefore, was not assayable.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Western Blot, Immu-Puri

Journal:

Article Title: The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling

doi: 10.1093/emboj/cdf345

Figure Lengend Snippet: Fig. 6. Model for the dynamics of mRNP structure in mammalian cells as a consequence of pre-mRNA capping, pre-mRNA splicing and mRNA export. In the nucleus, CBP80-bound pre-mRNA is spliced to generate CBP80-bound mRNA. This mRNA is bound by an EJC (dark gray shapes), consisting of RNPS1, Y14, SRm160 and REF/Aly, located 20–24 nucleotides upstream of the exon–exon junction. The EJC recruits the NMD factor Upf3/3X and the mRNA export factor TAP. Some mRNPs (possibly those subject to nucleus-associated NMD) undergo remodeling while associated with the nucleus (nucleus-associated remodeling). Other mRNPs (including those subject to cytoplasmic NMD) undergo remodeling after export to the cytoplasm (cytoplasmic remodeling). In both types of remodeling, CBP80 is replaced by eIF4E and the EJC is lost. Data indicate that the mostly perinuclear Upf2 protein is recruited by the EJC at a point when mRNA co-purifies with nuclei. In theory, recruitment could occur during mRNA export. However, if nucleus-associated remodeling takes place in the nucleoplasm, it is conceivable that an as yet undetected pool of Upf2 is nucleoplasmic. The single exon–exon junction exemplified is likely to typify all splicing-generated exon–exon junctions, regardless of position relative to the mRNA 3′ end.

Article Snippet: For all immunopurifications except those using anti-CTD antibody, samples were first cleared by incubation with end-over-end rotation in the presence of 50 µl of protein A–agarose beads (Boehringer Mannheim) in NET-2 buffer, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma), 2 mM benzamidine (Sigma) and, when RNase was not used, 100 U of RNase inhibitor (Promega) for 30 min at 4°C followed by centrifugation at 10 000 g . Cleared samples were then rotated in the presence of either normal rabbit antiserum (NRS; Gibco-BRL) or mouse IgG (Sigma), anti-CBP80 antibody ( Izaurralde et al., 1994 ), anti-eIF4E antibody ( Morley and McKendrick, 1997 ; Santa Cruz Biotechnology) or anti-hUpf3/3X antibody ( Ishigaki et al., 2001 ) for 90 min at 4°C, after which 2 mg of yeast RNA (Sigma) and protein A–agarose beads were added.

Techniques: Generated