Journal: Virology

Article Title: Enabled interferon signaling evasion in an immune-competent transgenic mouse model of parainfluenza virus 5 infection.

doi: 10.1016/j.virol.2007.10.001

Figure Lengend Snippet: Fig. 1. PIV5 V protein binds human but not mouse STAT2. Mouse NIH3T3 cells or human 2fTGH and HEK293Tcells were transfected with either FLAG-tagged PIV5 V protein (V) or FLAG vector control (C). Parallel samples of NIH3T3 were co-transfected with human STAT2 expression vector (hST2). Whole cell extracts were immunoprecipitated with FLAG-M2 agarose beads, eluted with SDS–PAGE buffer and processed for immunoblot with specific antibodies indicated.

Article Snippet: Phosphospecific antibodies against STAT2 and STAT4 as well as antibodies against murine STAT4 and the unique C-term region of human STAT2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used according to the manufacturer's instructions.

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Immunoprecipitation, SDS Page, Western Blot

Journal: Virology

Article Title: Enabled interferon signaling evasion in an immune-competent transgenic mouse model of parainfluenza virus 5 infection.

doi: 10.1016/j.virol.2007.10.001

Figure Lengend Snippet: Fig. 2. Expression of human STAT2 in transgenic mice. (A) The 2555-bp open reading frame encoding human STAT2 was flanked by the human ubiquitin C promoter on the 5′ end and an SV40 splice site and poly A tail on the 3′ end. (B) NIH3T3 cells were transfected with the 5xISRE-luciferase plasmid with or without the hSTAT2 transgene construct, infected with PIV5 for 24 h prior to luciferase assay. Normalized to co-transfected Renilla luciferase. Bars indicate the mean normalized to percent of maximum (n=3) and error bars indicate standard deviation. (C) Ubiquitous STAT2 expression in transgenic mice. Protein extracts (20 μg) from isolated transgenic mice were subject to immunoblot to detect human STAT2. Ubiquitous expression of the transgene throughout the mouse was observed.

Article Snippet: Phosphospecific antibodies against STAT2 and STAT4 as well as antibodies against murine STAT4 and the unique C-term region of human STAT2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used according to the manufacturer's instructions.

Techniques: Expressing, Transgenic Assay, Ubiquitin Proteomics, Transfection, Luciferase, Plasmid Preparation, Construct, Infection, Standard Deviation, Isolation, Western Blot

Journal: Virology

Article Title: Enabled interferon signaling evasion in an immune-competent transgenic mouse model of parainfluenza virus 5 infection.

doi: 10.1016/j.virol.2007.10.001

Figure Lengend Snippet: Fig. 3. Human STAT2 activity in mouse does not alter IFN signaling. (A) Splenocytes from transgenic and wild-type mice were treated with murine IFNβ for 5–30 min. Cells were immediately lysed and processed for immunoblot with antibodies that recognize human and mouse tyrosine phosphorylated STAT2, human STAT2, mouse STAT2 and STAT1α/β (recognizes human and mouse). (B) Splenocytes from transgenic and wild-type mice were treated with IFNβ for 10 min prior to lysate preparation and anti-STAT1 immunoprecipitation. Precipitated proteins were separated by SDS–PAGE and processed for immunoblot with antibodies for human STAT2, mouse STAT2 and tyrosine phosphorylated STAT2. (C) Splenocytes from hSTAT2 transgenic and wild-type mice were isolated and treated with mIFNβ for 6 or 18 h prior to RNA isolation and reverse transcription. Real-time PCR with primers specific for Mx1 and Ifi47 were performed and normalized to GAPDH. Graphs indicate average values for n=3, with error bars to represent standard deviation. (D) Transgenic and wild-type MEFs were pretreated 2 h with murine IFNβ, then infected with VSV (1 pfu/cell) for 16 h. Infectious virus released into the supernatant was estimated by titration on CV1 cells. IFN treatment provides a similar level of protection in transgenic and wild-type cells. Graph shows data from an individual VSV titration experiment. (E) Splenocytes were isolated from transgenic and wild-type mice and stimulated with IFNβ for 20′ prior to lysis. Whole cell extracts were separated by SDS–PAGE and immunoblotted with antibodies to detect STAT4 and tyrosine phosphorylated STAT4. IFNβ induces STAT4 activation in both wild-type and transgenic splenocytes.

Article Snippet: Phosphospecific antibodies against STAT2 and STAT4 as well as antibodies against murine STAT4 and the unique C-term region of human STAT2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used according to the manufacturer's instructions.

Techniques: Activity Assay, Transgenic Assay, Western Blot, Immunoprecipitation, SDS Page, Isolation, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, Infection, Virus, Titration, Lysis, Activation Assay

Journal: Virology

Article Title: Enabled interferon signaling evasion in an immune-competent transgenic mouse model of parainfluenza virus 5 infection.

doi: 10.1016/j.virol.2007.10.001

Figure Lengend Snippet: Fig. 5. Transgenic mouse cells support enhanced PIV5 replication. (A) MEFs were infected with PIV5 for 24 h before additional (1000 U/ml) exogenous IFNβ for another 24 h. Cells were lysed and processed for immunoblot with human STAT2 and P/Vantibodies. (B) PIV5 titer from MEFs infected at low MOI (1 pfu/cell) with PIV5 after 24 and 48 h. Viral supernatant was titered by serial dilution on CV-1 cells. Results show greater viral replication in transgenic MEFs.

Article Snippet: Phosphospecific antibodies against STAT2 and STAT4 as well as antibodies against murine STAT4 and the unique C-term region of human STAT2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used according to the manufacturer's instructions.

Techniques: Transgenic Assay, Infection, Western Blot, Serial Dilution

Journal: Molecular Biology of the Cell

Article Title: The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

doi: 10.1091/mbc.E16-06-0379

Figure Lengend Snippet: Schematic representation of known phosphorylation sites in human TTP family members. (A) NTD and CTD represent the N-terminal and C-terminal activation domains on either sides of the zinc-finger (ZF) RNA-binding domain (RBD). The serines that have been shown previously to regulate TTP family function upon phosphorylation are indicated in red. Corresponding serines in mouse proteins are indicated with brackets. The conserved C-terminal sequence between TIS11b and TIS11d is indicated between dotted lines. Other phosphoserines have been identified in vitro and in phosphoproteomic studies of ectopically expressed TTP (reviewed in ). However, the relevance of these phosphosites remains to be validated experimentally. No phosphorylated serines in TIS11d/BRF2 have been reported so far. (B) Protein kinases regulating TTP family fate and function. aa, amino acid.

Article Snippet: Polyclonal phosphospecific antibodies against TIS11b-phospho-S54 and TIS11b-phospho-S334 were generated by injection of the synthetic keyhole limpet hemocyanin–conjugated peptides CAGGGFPRRH(Sp)VTL or RRLPIFSRL(Sp)ISD, respectively, into rabbits (CovalAb, Lyon, France).

Techniques: Phospho-proteomics, Activation Assay, RNA Binding Assay, Sequencing, In Vitro

Journal: Molecular Biology of the Cell

Article Title: The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

doi: 10.1091/mbc.E16-06-0379

Figure Lengend Snippet: ACTH-induced expression of TIS11b mRNA in adrenocortical cells is transcription dependent. (A) Time-course analysis of TIS11 family member expression in BAC cells challenged with ACTH. RT-qPCR was performed as described in the Supplemental Material. Expression levels of TTP, TIS11b, and TIS11d mRNAs were normalized to RPL27 mRNA. Values are means ± SD of four independent experiments. (B) Northern blot analysis of TIS11b transcript levels in BAC cells challenged with ACTH in the absence or presence of the transcription inhibitor DRB. The membrane was hybridized to a radiolabeled TIS11b cDNA probe and rehybridized to 18S RNA probe for loading control. Shown is a representative Northern blot of six independent experiments.

Article Snippet: Polyclonal phosphospecific antibodies against TIS11b-phospho-S54 and TIS11b-phospho-S334 were generated by injection of the synthetic keyhole limpet hemocyanin–conjugated peptides CAGGGFPRRH(Sp)VTL or RRLPIFSRL(Sp)ISD, respectively, into rabbits (CovalAb, Lyon, France).

Techniques: Expressing, Quantitative RT-PCR, Northern Blot, Membrane, Control

Journal: Molecular Biology of the Cell

Article Title: The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

doi: 10.1091/mbc.E16-06-0379

Figure Lengend Snippet: Cloning and analysis of TIS11b promoter. (A) The sequence of the human TIS11b promoter region from −1670 nucleotides upstream of the transcription start site (+1) to the translation initiation ATG was determined using Ensembl Genome Browser ( www.ensembl.org ). The CRE is shown in red, and the TATAA box (bold) is underlined. (B) The CRE in TIS11b promoter is conserved among species. (C) Schematic representations of TIS11b promoter region constructs. The WT construct contains 1088 base pairs of the promoter region. The mutCRE construct results from the substitution of four nucleotides within the CREB consensus sequence as described in the Supplemental Material. (D) WT and mutCRE constructs were inserted in pGL3-luciferase reporter plasmid and transfected in COS7 cells as described in the Supplemental Material. Promoter-driven luciferase activity was measured after cell stimulation with 25 μM forskolin in the presence or absence of RpcAMP (10 nM) for 24 h. Results are represented as a percentage of luciferase activity in control nonstimulated cells. Transfections were performed in triplicate, and values are means ± SD from three independent experiments. *, significantly different from pWT luciferase activity in control nonstimulated cells, with p < 0.05; #, significantly different from pWT luciferase activity in forskolin-treated cells, with p < 0.05.

Article Snippet: Polyclonal phosphospecific antibodies against TIS11b-phospho-S54 and TIS11b-phospho-S334 were generated by injection of the synthetic keyhole limpet hemocyanin–conjugated peptides CAGGGFPRRH(Sp)VTL or RRLPIFSRL(Sp)ISD, respectively, into rabbits (CovalAb, Lyon, France).

Techniques: Cloning, Sequencing, Construct, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Cell Stimulation, Control

Journal: Molecular Biology of the Cell

Article Title: The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

doi: 10.1091/mbc.E16-06-0379

Figure Lengend Snippet: ACTH induces a cAMP-dependent expression and phosphorylation of TIS11b. (A) BAC cells were preincubated in the absence or presence of H89 (5 μM) for 30 min before addition of 10 nM of ACTH for the indicated periods of time. TIS11b and VEGF protein levels of whole-cell extracts (20 μg) were analyzed by Western blot. The blot was subsequently probed with an anti–β-actin to assess equal loading of samples. (B–D) Quantification of TIS11b, VEGF mRNA, and protein levels from independent experiments ( n = 5, means ± SEM). Protein-level values were normalized to actin and are expressed as percentage of control values at time 0 (unstimulated cells). VEGF mRNA levels were measured by quantitative PCR and normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT). (E) Time-course of TIS11b phosphorylation in BAC cells stimulated with 10 nM of ACTH in the presence of [ 32 P]orthophosphate and in the presence or absence of H89. TIS11b was immunoprecipitated (IP) from cell extracts, resolved by SDS–PAGE, and then visualized by autoradiography. One representative experiment of four is shown. (F) Quantification of phospho-TIS11b/total TIS11b ratio in ACTH-stimulated BAC cells ( n = 4, means ± SEM). (G) Phosphorylation of recombinant TIS11b by the catalytic subunit of PKA. Purified GST-TIS11b fusion protein was produced as described previously ( Ciais et al. , 2004 ). Increasing doses of GST-TIS11b were subjected to in vitro phosphorylation as described in Materials and Methods . Protein extract from Escherichia coli (30 μg) transformed with empty pGEX vector was used as control in the phosphorylation assay (first lane, 0 μg).

Article Snippet: Polyclonal phosphospecific antibodies against TIS11b-phospho-S54 and TIS11b-phospho-S334 were generated by injection of the synthetic keyhole limpet hemocyanin–conjugated peptides CAGGGFPRRH(Sp)VTL or RRLPIFSRL(Sp)ISD, respectively, into rabbits (CovalAb, Lyon, France).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Real-time Polymerase Chain Reaction, Immunoprecipitation, SDS Page, Autoradiography, Recombinant, Purification, Produced, In Vitro, Transformation Assay, Plasmid Preparation

Journal: Molecular Biology of the Cell

Article Title: The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

doi: 10.1091/mbc.E16-06-0379

Figure Lengend Snippet: S54 and S334 are PKA target sites in vitro and in vivo. (A) Sequence alignment of conserved amino acid within the N-terminus and the C-terminus between TIS11b, TIS11d, and TTP showing PKA consensus motifs (highlighted in red, RRHS and RLS). These motifs are also conserved between species in TIS11b sequence and harbor S54 S334 ( hs , Homo sapiens ; Bt , Bos taurus ; Rt , Rattus norvegicus ; mm , Mus musculus ; xl , Xenopus laevis ). (B) Dose-dependent in vitro phosphorylation of synthetic N-terminal and C-terminal peptides of TIS11b by the catalytic subunit of PKA. Both peptides contain the PKA consensus motifs RRHS or RLS (S54 and S334 are shown in bold; aa, amino acids). Phosphorylated peptides were resolved by SDS–PAGE (15%) and visualized by autoradiography. (C) In vitro phosphorylation of recombinant Flag-WT TIS11b and Flag-TIS11b mutants S54A and S334A (1 μg purified proteins). Protein extracts from E. coli transformed with empty vector (pET15b) served as control (Vect). (D) PKA-mediated phosphorylation of recombinant TIS11b was significantly impaired when S54 and S334 were replaced by an alanine. Ratios of phosphorylated protein/total protein are reported ( n = 4 independent experiments, mean ± SEM). Asterisks: significantly different from the WT with ** p < 0.01 and *** p < 0.001. (E) Characterization of the phosphospecific antibodies in vitro. Unphosphorylated control peptides were run alongside phosphorylated peptides to determine whether the antibodies could detect the phospho-S34 (pS54) or phospho-S334 (pS334). (F) Characterization of the phosphospecific antibodies in forskolin-stimulated cells. COS7 cells were transfected with empty pTarget Vector (Vect), pTarget-WT TIS11b (WT), pTarget-TIS11b-S54A, or pTarget-TIS11b-S334A plasmids and then stimulated or not (basal) with 25 μM of forskolin for 60 min in the presence or absence of H89. Cell lysates were analyzed by Western blot to assess the specificity of the anti-pS54 or anti-pS334 antibodies. Blots were imaged simultaneously with the Chemidoc Imaging system for 5 s to accurately detect strong and weak bands for all conditions.

Article Snippet: Polyclonal phosphospecific antibodies against TIS11b-phospho-S54 and TIS11b-phospho-S334 were generated by injection of the synthetic keyhole limpet hemocyanin–conjugated peptides CAGGGFPRRH(Sp)VTL or RRLPIFSRL(Sp)ISD, respectively, into rabbits (CovalAb, Lyon, France).

Techniques: In Vitro, In Vivo, Sequencing, Phospho-proteomics, SDS Page, Autoradiography, Recombinant, Purification, Transformation Assay, Plasmid Preparation, Control, Transfection, Western Blot, Imaging

Journal: Molecular Biology of the Cell

Article Title: The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

doi: 10.1091/mbc.E16-06-0379

Figure Lengend Snippet: TIS11b is phosphorylated at S54 and S334 in hormone- and hypoxia-stimulated cells. (A) BAC cells were stimulated with ACTH in the presence or absence of H89 for the indicated periods of time. Total-cell extracts were probed for total TIS11b/TIS11d, pS54-TIS11b, and pS334-TIS11b. Owing to the conserved PKA consensus motifs (RRHS and RHS; see ) within TIS11b and TIS11d sequences, both phosphoproteins were detected. Note the shift of TIS11b toward high-molecular-weight species. The asterisk indicates the phospho-(S334)-TIS11b species appearing at 6 h poststimulation by ACTH. (B) A549 lung carcinoma cells were exposed to normoxia (0 h) or hypoxia for 2, 4, and 8 h. Cells extracts were probed by Western blot as described in A. (C) A549 cells were exposed to normoxia or hypoxia for 8 h in the absence or presence of OA, an inhibitor of the phosphatases PP2A/PP1. Note the pS334-TIS11b species appearing under hypoxia in the presence of OA (indicated by an asterisk). Blots were subsequently probed with an anti–β-actin to assess equal loading of samples.

Article Snippet: Polyclonal phosphospecific antibodies against TIS11b-phospho-S54 and TIS11b-phospho-S334 were generated by injection of the synthetic keyhole limpet hemocyanin–conjugated peptides CAGGGFPRRH(Sp)VTL or RRLPIFSRL(Sp)ISD, respectively, into rabbits (CovalAb, Lyon, France).

Techniques: High Molecular Weight, Western Blot

Journal: Molecular Biology of the Cell

Article Title: The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

doi: 10.1091/mbc.E16-06-0379

Figure Lengend Snippet: S54 and S334 regulate TIS11b protein stability. (A) COS7 cells were cotransfected with 10 ng of pTarget plasmids encoding WT TIS11b (WT), TIS11b S54A, TIS11b S54D, TIS11b S334A, or TIS11b S334D mutants and then treated with cycloheximide (CHX) as described in Materials and Methods . The half-life of WT or mutant TIS11b was analyzed by Western blot. All the membranes were exposed simultaneously for 15 s. Quantification of TIS11b steady-state levels (TIS11b/actin ratio) at t = 0 yielded the ratios 1, 0.61, 1.27, 0.85, and 1.58 for WT TIS11b, TIS11b-S54A, S54D, TIS11b-S334A, and TIS11b-S334D mutants, respectively. (B) TIS11b protein levels were normalized to actin levels and plotted as a percentage of the initial value against time using nonlinear regression to a first-order exponential decay model. (C) Calculated half-lives of TIS11b from four independent experiments. Half-lives of TIS11b mutants were compared with the half-life of WT TIS11b. p Values and 95% confidence intervals (CIs) were determined using an F -test.

Article Snippet: Polyclonal phosphospecific antibodies against TIS11b-phospho-S54 and TIS11b-phospho-S334 were generated by injection of the synthetic keyhole limpet hemocyanin–conjugated peptides CAGGGFPRRH(Sp)VTL or RRLPIFSRL(Sp)ISD, respectively, into rabbits (CovalAb, Lyon, France).

Techniques: Mutagenesis, Western Blot

Journal: Molecular Biology of the Cell

Article Title: The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

doi: 10.1091/mbc.E16-06-0379

Figure Lengend Snippet: The phosphomimetic TIS11b-S334D mutant is more potent than WT TIS11b in decreasing VEGF 3′ UTR–driven luciferase activity and endogenous VEGF mRNA steady-state levels. (A) COS7 cells were cotransfected with pLuc 3′ UTR and pTarget plasmids encoding WT TIS11b, TIS11b-S54A, TIS11b-S54D, TIS11b-S334A, or TIS11b-S334D mutants. Firefly/ Renilla luciferase activities of cell lysates were measured as described in Materials and Methods . Results are expressed as relative light units of firefly luciferase activity over relative light units of Renilla luciferase activity and are represented as a percentage of the luciferase activity in control cells transfected with empty pTarget plasmid. Transfections were performed in triplicate, and values are means ± SEM from seven independent experiments. The lower panel is a representative Western blot analysis of overexpressed TIS11b proteins showing that equivalent amounts of overexpressed TIS11b were recovered. (B) Northern blot (NB) analysis of endogenous VEGF mRNA in COS7 cells transfected as in A. (C) Western blot (WB) analysis of TIS11b protein expression levels in the COS7 cells used for the Northern experiment shown in B. (D) Quantification of VEGF mRNA steady-state levels in four independent experiments (means ± SEM). Asterisks: significantly different from WT with * p < 0.05 and ** p < 0.01 (one-way ANOVA).

Article Snippet: Polyclonal phosphospecific antibodies against TIS11b-phospho-S54 and TIS11b-phospho-S334 were generated by injection of the synthetic keyhole limpet hemocyanin–conjugated peptides CAGGGFPRRH(Sp)VTL or RRLPIFSRL(Sp)ISD, respectively, into rabbits (CovalAb, Lyon, France).

Techniques: Mutagenesis, Luciferase, Activity Assay, Control, Transfection, Plasmid Preparation, Western Blot, Northern Blot, Expressing

Journal: Molecular Biology of the Cell

Article Title: The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

doi: 10.1091/mbc.E16-06-0379

Figure Lengend Snippet: VEGF mRNA is more efficiently destabilized by the phosphomimetic TIS11b S334D mutant. (A) COS7 cells were transfected in 12-well plates with pTarget empty plasmid (Vector) or plasmids encoding WT or mutant TIS11b. AT 48 h after transfection, the transcription inhibitor DRB (10 μg/ml) was added, and total RNA was extracted at the indicated time points and analyzed by Northern blot. The membrane was hybridized to a radiolabeled VEGF 3′ UTR probe and rehybridized to 18S RNA probe for loading control. (B) VEGF mRNA levels were normalized to 18s RNA levels and plotted as a percentage of the initial value against time using nonlinear regression to a first-order exponential decay model. Shown are the mRNA decay rates from three pooled independent experiments. (C) VEGF mRNA half-lives were calculated and compared with the half-life of the transcript in the presence of WT TIS11b. p Values and 95% CIs were determined using an F -test.

Article Snippet: Polyclonal phosphospecific antibodies against TIS11b-phospho-S54 and TIS11b-phospho-S334 were generated by injection of the synthetic keyhole limpet hemocyanin–conjugated peptides CAGGGFPRRH(Sp)VTL or RRLPIFSRL(Sp)ISD, respectively, into rabbits (CovalAb, Lyon, France).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Northern Blot, Membrane, Control

Journal: Molecular Biology of the Cell

Article Title: The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

doi: 10.1091/mbc.E16-06-0379

Figure Lengend Snippet: TIS11b S334D mutant exhibits enhanced interaction with the decapping complex activator Dcp1a. (A) Sequence alignment of the distal C-terminus of TTP family members, showing that the TTP-CCR4-NOT interaction motif is highly conserved between TTP family members ( Fabian et al. , 2013 ). (B) Western blots showing association of TIS11b proteins with endogenous mRNA decay enzymes. Transiently expressed WT or TIS11b mutants were immunoprecipitated from HEK 293T cell extracts, and precipitates were probed for the presence of TIS11b and mRNA decay proteins. The mRNA-stabilizing protein HuR served as negative control and was not associated with TIS11b. NRS: immunoprecipitation reaction using normal rabbit serum in place of TIS11b antibodies. Input: 5% total-cell extract. (C, D) Quantification of Cnot1/TIS11b and Dcp1a/TIS11b ratio in three independent immunoprecipitation experiments. *, Significantly different from WT TIS11b with p < 0.05; **, significantly different from S334A mutant with p < 0.01 (one-way ANOVA).

Article Snippet: Polyclonal phosphospecific antibodies against TIS11b-phospho-S54 and TIS11b-phospho-S334 were generated by injection of the synthetic keyhole limpet hemocyanin–conjugated peptides CAGGGFPRRH(Sp)VTL or RRLPIFSRL(Sp)ISD, respectively, into rabbits (CovalAb, Lyon, France).

Techniques: Mutagenesis, Sequencing, Western Blot, Immunoprecipitation, Negative Control

Journal: Cancer Research

Article Title: Isoleucine, an Essential Amino Acid, Prevents Liver Metastases of Colon Cancer by Antiangiogenesis

doi: 10.1158/0008-5472.can-06-3739

Figure Lengend Snippet: Figure 4. A and B, effects of isoleucine and leucine on VEGF mRNA levels in vivo. After they were pretreated with isoleucine or leucine (3 mg/d) for 5 d, colon 26 cells (1 104) were implanted into the spleens of BALB/c mice. Three weeks later, mice were sacrificed and their livers were removed for further studies. Representative gel from RT-PCR of VEGF mRNA (n = 5 each group) in mice liver. Lane 1, liver of isoleucine-treated mice (nontumor tissue); lane 2, liver of leucine-treated mice (nontumor tissue); lane 3, liver metastatic lesion from leucine-treated mice; lane 4, liver from PBS-treated mice (without colon 26 challenge); lane 5, spleen from isoleucine-treated mice (without colon 26 challenge); lane 6, spleen from leucine-treated mice (without colon 26 challenge). *, P < 0.05; **, P < 0.01. C, representative Western blot of HIF1-a and phosphorylated 4E-BP1 and Stat3 proteins in mouse liver. Lane 1, isoleucine-treated mice (nontumor liver); lane 2, leucine-treated mice (nontumor liver); lane 3, cancer lesion from leucine-treated mouse; lane 4, liver from PBS-treated mice (without colon 26 challenge).

Article Snippet: After blocking, the membranes were incubated overnight at 4jC with antibodies against hypoxiainducible factor 1-a (HIF1-a; Santa Cruz Biotechnology, Santa Cruz, CA) or phosphospecific antibodies against 4E-BP1 (Thr37/46; Cell Signaling Technology, San Diego, CA) and signal transducers and activators of transcription 3 (Stat3; Santa Cruz Biotechnology).

Techniques: In Vivo, Reverse Transcription Polymerase Chain Reaction, Western Blot