Journal: Journal of Biological Chemistry

Article Title: Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

doi: 10.1074/jbc.m112.369405

Figure Lengend Snippet: FIGURE 2. Structural determinants of NHERF1 association with Npt2a. A, OKH cells were transiently transfected with empty vector (control), FLAG-NHERF1, HA-GFP-Npt2a or HA-GFP-Npt2a(L639A). 48 h after transfection, the cells were treated with vehicle or 100 nM PTH(1–34) for 2 h. FLAG-tagged proteins were immunoprecipitated with FLAG-agarose. The precipitated protein was then immunoblotted with HA antibody. Actin expression was used as a loading control. The figure is representative of three independent experiments. B, Npt2a interacts with PDZ1. OKH-HA-GFP-Npt2a cells were transiently transfected with empty vector, wild-type NHERF1, or NHERF1 harboring mutations in PDZ1 and/or PDZ2 core-binding domains (sPDZ1-NHERF1, sPDZ2-NHERF1, and sPDZ1/2- NHERF1) or truncated NHERF1 lacking the ezrin-binding domain (NHERF1-EBD). 48 h after transfection, HA-tagged proteins were immunoprecipitated with HA-agarose. The precipitated protein was then immunoblotted with NHERF1 antibody. An example of three independent experiments is shown. C, sodium- dependent Pi uptake was measured in OKH cells transiently transfected with empty vector, wild-type NHERF1, sPDZ1-NHERF1, sPDZ2-NHERF1, sPDZ1/2- NHERF1, or NHERF1-EBD and treated with vehicle or PTH(1–34) (100 nM, 2 h). Data are summarized as the mean S.E. (error bars) (n 3; **, p 0.01, versus vector). D, OKH cells were transfected with HA-PTHR, FLAG-NHERF1, FLAG-L110V-NHERF1, FLAG-R153Q-NHERF1, or FLAG-E225K-NHERF1. 48 h after transfec- tion,FLAG-taggedproteinswereimmunoprecipitated(IP).Theprecipitatedproteinwasimmunoblotted(IB)withHAantibody.Dataarerepresentativeofthree independent experiments. E, OKH cells were transfected with empty vector, wild-type NHERF1, or mutated NHERF1. Cell surface binding of [125I]PTH(1–34) was measured as described under “Experimental Procedures.” Data are summarized as the mean S.E. of triplicate determinations. F, OKH cells were transfected with wild-type NHERF1 or mutated forms of NHERF1. Cells were treated with 100 nM PTH for 15 min, and cAMP accumulation was measured as described under “Experimental Procedures.” Data are summarized as the mean S.E. (n 4). IP, immunoprecipitation; IB, immunoblot.

Article Snippet: The cleaved, phosphorylated NHERF1 fragments were resolved on 16.5% Tricine-SDS-PAGE (Bio-Rad).

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, Expressing, Binding Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

doi: 10.1074/jbc.m112.369405

Figure Lengend Snippet: FIGURE 3. NHERF1 forms a ternary complex with Npt2a and ezrin. A, Npt2a, NHERF1, and ezrin form a ternary complex (lane 2) that dissoci- ates upon treatment with PTH (lane 6). Truncated NHERF1 lacking the EBD (NHERF1-EBD) and Npt2a in which the carboxyl-terminal PDZ-binding domain was mutated (Npt2a(L639A)) do not form a ternary complex. B, mutant NHERF1 constructs are poorly able to form a ternary complex with Npt2a and ezrin and are refractory to PTH. Shown is a representative result of OKH cells stably expressing HA-Npt2a transiently transfected with FLAG-tagged wild-type NHERF1 or L110V-, R153Q-, or E225K-NH- ERF1. 48 h after transfection, the cells were treated, and the ternary com- plex was detected as above. C, aggregate results from experiments shown in B (n 3; **, versus no PTH, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple-comparison test)).

Article Snippet: The cleaved, phosphorylated NHERF1 fragments were resolved on 16.5% Tricine-SDS-PAGE (Bio-Rad).

Techniques: Binding Assay, Mutagenesis, Construct, Stable Transfection, Expressing, Transfection, Comparison

Journal: Journal of Biological Chemistry

Article Title: Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

doi: 10.1074/jbc.m112.369405

Figure Lengend Snippet: FIGURE 5. NHERF1 mutants are resistant to phosphorylation upon PTH stimulation. A, PTH promotes phosphorylation of wild-type NHERF1 but not of NHERF1 mutants. Representative experiment shows PTH-stimulated phos- phorylation within the two large fragments of NHERF1 cleaved by CNBr con- taining Ser77 within PDZ1 (residues 2–156) and in a Ser-rich cluster located in the linker between PDZ2 and the EBD (residues 208–329) (Cluster). B, quanti- fication of phosphorylation results from A (n 3; **, PTH versus control, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple- comparison test)). Error bars, S.E.; IB, immunoblot.

Article Snippet: The cleaved, phosphorylated NHERF1 fragments were resolved on 16.5% Tricine-SDS-PAGE (Bio-Rad).

Techniques: Phospho-proteomics, Control, Comparison, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

doi: 10.1074/jbc.m112.369405

Figure Lengend Snippet: FIGURE 6. Functional and structural analysis of NHERF1 mutations. A and B, R153Q-NHERF1 acts as a loss-of-function mutation and not as a dominant negative inhibitor of wild-type NHERF1. OKH cells were transfected with 0.2 g of GFP-NHERF1 alone or 0–1.0 g of R153Q-NHERF1, with the balance made of empty vector (A). PTH-sensitive Pi uptake was measured as before (B) (n 3 independent determinations). C and D, affinity of wild-type NHERF1 (WT) and mutant R153Q-NHERF1 for Npt2a was determined by fluorescence polarization, and KD was derived from the calculated anisotropy (C). Affinity measurements by ITC for wild-type NHERF1 (D, left) and R153Q-NHERF1 (D, right) are shown. Error bars, S.E.; IB, immunoblot.

Article Snippet: The cleaved, phosphorylated NHERF1 fragments were resolved on 16.5% Tricine-SDS-PAGE (Bio-Rad).

Techniques: Functional Assay, Mutagenesis, Dominant Negative Mutation, Transfection, Plasmid Preparation, Fluorescence, Derivative Assay, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

doi: 10.1074/jbc.m112.369405

Figure Lengend Snippet: FIGURE 7. Rescue of NHERF1 structure and function. A, rescue of loss of function of R153-NHERF1 mutant by preventing closed NHERF1 conformation. PTH-sensitive Pi uptake was measured in OKH cells transfected with the indicated construct. Results are the average S.E. (error bars) (n 4; **, versus vector, p 0.001, one-way repeated measures ANOVA (Dunnett’s post hoc multiple-comparison test)). B–E, double R153Q/L358A-NHERF1 mutation restores PTH- induced phosphorylation to NHERF1 (B and C) (n 3; **, PTH versus control, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple- comparison test)) and inhibition by H89 and Ht31 of PTH-sensitive Pi uptake (D) (n 3; **, PTH versus St-Ht31 PTH, p 0.001, two-way repeated measures ANOVA (Bonferroni post hoc multiple-comparison test)) and reestablishes the ability to form the ternary complex even in the presence of the R153Q mutation (E). IP, immunoprecipitation; IB, immunoblot.

Article Snippet: The cleaved, phosphorylated NHERF1 fragments were resolved on 16.5% Tricine-SDS-PAGE (Bio-Rad).

Techniques: Mutagenesis, Transfection, Construct, Plasmid Preparation, Comparison, Phospho-proteomics, Control, Inhibition, Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Ezrin-anchored Protein Kinase A Coordinates Phosphorylation-dependent Disassembly of a NHERF1 Ternary Complex to Regulate Hormone-sensitive Phosphate Transport

doi: 10.1074/jbc.m112.369405

Figure Lengend Snippet: FIGURE 8. Model of PTH-mediated disassembly of the Npt2a-NHERF1-ezrin ternary complex and endocytosis of Npt2a. A, in the resting state, Npt2a is presentinbrushbordersofapicalmembranes,boundtoPDZ1ofwild-typeNHERF1aspartofthespontaneouslyformedNpt2a-NHERF1-ezrinternarycomplex. The EBD of NHERF1 binds to ezrin, linking the complex to cytoskeletal elements. In this way, Npt2a is tethered to apical membranes and mediates phosphate uptake. Upon PTH exposure, PTHR stimulates cAMP formation and activates PKA. PKA regulatory (R) subunits undergo a conformational change and release the catalytic (C) subunits. Ezrin binds to PKA regulatory subunits, positioning them in close proximity to the ternary complex. PKA catalytic subunits phosphor- ylate (P) NHERF1, with subsequent dissociation of Npt2a, which is endocytosed, thereby inhibiting phosphate transport. B, in the presence of mutant forms of NHERF1, less Npt2a is present in brush border apical membranes. PTH stimulates cAMP normally. Because NHERF1 is locked in an inactive conformation, access to catalytic PKA subunits is prevented, thereby precluding phosphorylation and dissociation of tethered Npt2a from NHERF1.

Article Snippet: The cleaved, phosphorylated NHERF1 fragments were resolved on 16.5% Tricine-SDS-PAGE (Bio-Rad).

Techniques: Mutagenesis, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: CD40 Induces Interleukin-6 Gene Transcription in Dendritic Cells

doi: 10.1074/jbc.m109250200

Figure Lengend Snippet: FIG. 1. Induction of cytokines in FSDCs and BMDCs during culture with anti-CD40 mAb 3/23. mRNA was isolated from FSDCs or BMDCs which were either incubated with 30 g/ml anti-CD40 mAb 3/23 or left untreated for 24 h. The mRNA was used to obtain first strand cDNA, which was used as a template in RT-PCR reactions using protocols described under “Materials and Methods.” Utilizing this method, cDNA species encoding murine IL-6, IL-12 (p35 and p40), IFN, IL-4, and -actin were amplified. For IFN and IL-4, positive control RT-PCR reactions are shown on the far left-hand side and used mRNA extracted from total blood lymphocytes (TBL) taken from a Balb/c mouse. The gels shown are representative of at least two inde- pendent experiments. A 1-kilobase DNA ladder was run alongside the PCR products to confirm correct sizes of the amplified cDNA fragments.

Article Snippet: Mouse anti-human CD40 monoclonal antibody LOB7.6 was obtained from Serotec, UK.

Techniques: Isolation, Incubation, Reverse Transcription Polymerase Chain Reaction, Amplification, Positive Control

Journal: Journal of Biological Chemistry

Article Title: CD40 Induces Interleukin-6 Gene Transcription in Dendritic Cells

doi: 10.1074/jbc.m109250200

Figure Lengend Snippet: FIG. 2. Induction of IL-6 promoter activity by anti-CD40 and requirement for specific regulatory DNA motifs. A, FSDCs were transfected with 100 ng of pRLTK and 1 g of either wild type pIL6- Luc651 or mutated pIL6-Luc651 constructs carrying mutation in the AP1, NF-B, and NF-IL6 sites. The transfected cultures were split into two flasks. 24 h later, one culture flask was incubated with 30 g/ml anti-CD40 mAb 3/23 for a further 24 h, while the remaining flask was left untreated. Samples treated with 3/23 are depicted in black solid boxes, whereas untreated samples are in white. Luciferase activities were normalized to pRLTK activity and expressed as the mean S.E. of three independent transfection experiments. Statistical analysis was performed by Student’s t test. *, **, and *** denote p 0.05, 0.01, and 0.005, respectively. B, fold induction of the sample incubated with anti-CD40 mAb 3/23 versus the non-treated sample for each of the pIL6-Luc651 constructs.

Article Snippet: Mouse anti-human CD40 monoclonal antibody LOB7.6 was obtained from Serotec, UK.

Techniques: Activity Assay, Transfection, Construct, Mutagenesis, Incubation, Luciferase

Journal: Journal of Biological Chemistry

Article Title: CD40 Induces Interleukin-6 Gene Transcription in Dendritic Cells

doi: 10.1074/jbc.m109250200

Figure Lengend Snippet: FIG. 3. Induction of the IL-6 pro- moter requires activation of NF-B. A, FSDCs were transfected with 100 ng of pRLTK, 1 g of wild type pIL6-Luc651, and 2 g of IB trans-dominant negative (IBD) expression vector or pcDNA3 as control. The transfected cultures were split into two flasks. 24 h after transfec- tion, one flask was incubated with 30 g/ml anti-CD40 mAb 3/23 for 24 h while the remaining flask was left untreated. Luciferase activity was determined 48 h after transfection. Luciferase activities were normalized to pRLTK activity and expressed as the mean S.E. of three independent transfection experiments. Samples treated with 3/23 are depicted in black solid boxes, whereas untreated sam- ples are in white. Statistical analysis was performed by Student’s t test. *** denotes p 0.005. B, fold induction of the samples incubated with anti-CD40 mAb 3/23 ver- sus the non-treated samples as shown in Fig. 3A. C, mRNA was isolated from BM- DCs which were either untreated () or incubated for 24 h with 30 g/ml anti- CD40 mAb 3/23 together with either 5 M MG132 dissolved in Me2SO or Me2SO alone. The mRNA was used to obtain first strand cDNA, which was used as a tem- plate in RT-PCR. cDNA species encoding murine IL-6 and -actin were amplified over 32 and 27 cycles in RT-PCR reactions using protocols described under “Materi- als and Methods.” The gels shown are rep- resentative of at least two independent experiments. A 1-kilobase DNA ladder was run alongside the PCR products to confirm correct sizes of the amplified cDNA fragments.

Article Snippet: Mouse anti-human CD40 monoclonal antibody LOB7.6 was obtained from Serotec, UK.

Techniques: Activation Assay, Transfection, Dominant Negative Mutation, Expressing, Plasmid Preparation, Control, Incubation, Luciferase, Activity Assay, Isolation, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: Journal of Biological Chemistry

Article Title: CD40 Induces Interleukin-6 Gene Transcription in Dendritic Cells

doi: 10.1074/jbc.m109250200

Figure Lengend Snippet: FIG. 4. EMSA analysis of NF-B and CBF1 in anti-CD40 stimulated FSDC. A, nuclear extracts from control or FSDC cells treated with anti-mCD40 mAb 3/23 for 24 h were isolated and 2 g used in EMSA with NF-B double stranded oligonucleotide probe. Two (1 and 2) specific DNA-protein complexes were assembled and are denoted by arrows. Supershift analysis was performed on control and treated samples using antisera recognizing p50 and p65 or JunB as a control. B, 2 g of nuclear extracts from FSDC cells treated with anti-mCD40 mAb 3/23 for 24 h (as obtained in A) were incubated with double stranded NF-B oligonucleotide probe, or NF-B probe lacking p50/p65 or CBF1-binding sites. The nucleotide substitutions introduced into the wild type NF-B oligonucleotide to generate mutant oligonucleotides lacking p50/p65 or CBF1-binding sites are shown below the EMSA gel. C, 2 g of nuclear extracts from FSDC cells treated with anti-mCD40 mAb 3/23 for 24 h (as obtained in A) were incubated with double stranded NF-B oligonucleotide probe and supershift analysis performed using antisera recognizing p50, p65, CBF1, or JunB as a control.

Article Snippet: Mouse anti-human CD40 monoclonal antibody LOB7.6 was obtained from Serotec, UK.

Techniques: Control, Isolation, Incubation, Binding Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: CD40 Induces Interleukin-6 Gene Transcription in Dendritic Cells

doi: 10.1074/jbc.m109250200

Figure Lengend Snippet: FIG. 5. Induction of AP-1 activity by CD40 ligation. A, nuclear extracts from control or FSDC cells treated with anti-mCD40 mAb 3/23 for 16 h were isolated and 10 g used in EMSA with AP1 double stranded oligonucleotide probe in presence of 100 M excess of unlabeled nonspecific oligonucleotide (Sp1) or unlabeled specific AP1 oligonucleotide. B, supershift analysis was performed on 3/23 mAb treated FSDC samples using antisera recognizing c-Jun, JunD, JunB, c-Fos, or Sp1 as a control. Supershift complexes are shown for extracts incubated with JunB and JunD antisera. Asterisks placed to the left of the supershift complexes are included to aid identification of these species. C, immunoblot analysis of JunD, JunB, and c-Jun protein expression was performed on crude cytoplasmic and nuclear extracts from control and FSDCs treated with 3/23 mAb for 16 h. All gels are representative of three independent experiments. D, FSDCs were transfected with 100 ng of pRLTK, 1 g of wild type pIL6-Luc651, and 2 g of JunD dominant negative expression vector pRSV-JunD or pRSV as control. The transfected cultures were split into two flasks and after 24 h one flask was incubated with 30 g/ml anti-CD40 mAb 3/23 while the remaining flask was left untreated. Luciferase activity was determined 48 h after transfection. Luciferase activities were normalized to pRLTK activity and expressed as the mean S.E. of three independent transfection experiments. Samples treated with 3/23 are depicted in black solid boxes, whereas untreated samples are in white. Statistical analysis was performed by Student’s t test. ** denotes p 0.01.

Article Snippet: Mouse anti-human CD40 monoclonal antibody LOB7.6 was obtained from Serotec, UK.

Techniques: Activity Assay, Ligation, Control, Isolation, Incubation, Western Blot, Expressing, Transfection, Dominant Negative Mutation, Plasmid Preparation, Luciferase

Journal: Journal of Biological Chemistry

Article Title: CD40 Induces Interleukin-6 Gene Transcription in Dendritic Cells

doi: 10.1074/jbc.m109250200

Figure Lengend Snippet: FIG. 6. Requirement for specific amino acid sequences in the cytoplasmic domain of CD40. A, 1 g of wild type pIL6–651Luc was transfected into FSDC cells along with 100 ng of pRLTK and 2 g of empty vector pcDNA3 or pcDNA3-derived expression vectors carrying cDNA cassettes for wild type hCD40, truncated hCD40 containing three remaining intracellular residues (hCD40KKV), hCD40 mutants carrying point mutations (hCD40T254A, hCD40T254E, and hCD40T254S), hCD40 with a point mutation and a deletion of carboxyl-terminal 15 amino acids (hCD40T254A262), hCD40 with 15 carboxyl-terminal amino acids deleted (hCD40262), or a fusion protein made up of hCD450 extracellular and transmembrane domains and hCD22 intracellular domain. The transfected cultures were split into two flasks, one flask was treated with 300 g/ml anti-human CD40 mAb LOB7.6 for 24 h and the other was left untreated. 24 h later, cells were harvested and luciferase assays performed. Samples treated with LOB7.6 are depicted in black solid boxes, whereas untreated samples are in white. Luciferase activities were normalized to pRLTK activity and expressed as the mean S.E. of six independent transfection experiments. B, FSDC cells were transfected with 1 g of IB-Luc, 100 ng of pRLTK and hCD40 expression vectors and treated as already described in Fig. 5A. Luciferase activities were normalized to pRLTK activity and expressed as the means S.E. of five independent transfection experiments.

Article Snippet: Mouse anti-human CD40 monoclonal antibody LOB7.6 was obtained from Serotec, UK.

Techniques: Transfection, Plasmid Preparation, Derivative Assay, Expressing, Mutagenesis, Luciferase, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: CD40 Induces Interleukin-6 Gene Transcription in Dendritic Cells

doi: 10.1074/jbc.m109250200

Figure Lengend Snippet: FIG. 7. Inhibition of anti-CD40 induced IL-6 promoter activity by dominant negative TRAF2. A, mRNA was isolated from FSDCs which were either incubated with 30 g/ml anti-CD40 mAb 3/23, or left untreated for 24 h. The mRNA was used to obtain first strand cDNA, which was used as a template in RT-PCR reactions using protocols described under “Materials and Methods.” Utilizing this method, cDNA species encoding murine TRAF2, TRAF6, and -actin were amplified over 35, 35, and 28 PCR cycles, respectively. The gels shown are representative of at least two independent experiments. A 1-kilobase DNA ladder was run alongside the PCR products to confirm correct sizes of the amplified cDNA fragments. B, FSDCs were transfected with 100 ng of pRLTK, 1 g of IB-Luc, and 2 g of TRAF2 dominant negative (TRAF2 DN) expression vector or empty control vector. The cultures were split into 2 and 24 h after the transfection one-half was incubated with 30 g/ml anti-CD40 mAb 3/23 for 24 h. Luciferase activity was determined 48 h after transfection. C, FSDCs were transfected with 100 ng of pRLTK, 1 g of IB-Luc, and 2 g of TRAF6 dominant negative (TRAF6 DN) and processed as described for B. For both B and C, luciferase activities were normalized to pRLTK activity and expressed as the mean S.E. of three independent transfection experiments. Samples treated with 3/23 are depicted in black solid boxes, whereas untreated samples are in white. Statistical analysis was performed by Student’s t test. *** denotes p 0.005.

Article Snippet: Mouse anti-human CD40 monoclonal antibody LOB7.6 was obtained from Serotec, UK.

Techniques: Inhibition, Activity Assay, Dominant Negative Mutation, Isolation, Incubation, Reverse Transcription Polymerase Chain Reaction, Amplification, Transfection, Expressing, Plasmid Preparation, Control, Luciferase

Journal: Journal of Biological Chemistry

Article Title: CD40 Induces Interleukin-6 Gene Transcription in Dendritic Cells

doi: 10.1074/jbc.m109250200

Figure Lengend Snippet: FIG. 8. Induction of IL-6 gene transcription by anti-CD40 is a transient response. mRNA was isolated from control or FSDCs treated with anti-mCD40 mAb 3/23 for 24, 48, or 72 h. mRNA was used to obtain first strand cDNA which was used as a template in RT-PCR reactions using protocols described under “Materials and Methods.” Utilizing this method, cDNA species encoding murine IL-6 and -actin were amplified. The gels shown are representative of at least two independent experiments. A 1-kilobase DNA ladder was run alongside the PCR products to confirm correct sizes of the amplified cDNA fragments.

Article Snippet: Mouse anti-human CD40 monoclonal antibody LOB7.6 was obtained from Serotec, UK.

Techniques: Isolation, Control, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: Journal of Biological Chemistry

Article Title: CD40 Induces Interleukin-6 Gene Transcription in Dendritic Cells

doi: 10.1074/jbc.m109250200

Figure Lengend Snippet: FIG. 9. Prolonged induction of CBF1 DNA binding activity and repression of IL-6 promoter function. A, 2 g of nuclear extract isolated from FSDC cells treated with anti-mCD40 mAb 3/23 for 0, 2, 4, 6, 16, 24, and 48 h was used in EMSA with NF-B double stranded oligonucleotide probe. B, FSDCs were transfected with 100 ng of pRLTK, 1 g of wild type pIL6-Luc651, and 2 g of CBF1 expression vector pJH282 or empty vector pSG5. The cultures were split into two flasks and 24 h after transfection one flask was incubated with 30 g/ml anti-CD40 mAb 3/23 for 24 h, while the remaining flask was left untreated. The cells were then harvested and luciferase assay performed. Samples treated with 3/23 are depicted in black solid boxes, whereas untreated samples are in white. Luciferase activities were normalized to pRLTK activity and expressed as the mean S.E. of three independent transfection experiments. Statistical analysis was performed by Student’s t test. *** denotes p 0.005.

Article Snippet: Mouse anti-human CD40 monoclonal antibody LOB7.6 was obtained from Serotec, UK.

Techniques: Binding Assay, Activity Assay, Isolation, Transfection, Expressing, Plasmid Preparation, Incubation, Luciferase

Journal: Journal of Biological Chemistry

Article Title: CD40 Induces Interleukin-6 Gene Transcription in Dendritic Cells

doi: 10.1074/jbc.m109250200

Figure Lengend Snippet: FIG. 10. Conservation of CD40 signaling events between two distinct mouse DC lines (FSDC and DC2.4). To confirm the generality of findings with FSDC a series of key experiments were repeated using the mouse DC line DC2.4. DC2.4s were transfected with 100 ng of pRLTK, 1 g of wild type pIL6-Luc651wt; A, 2 g of IB dominant negative (IB DN) expression vector or pcDNA3 empty vector as a control; B, 2 g of JunD dominant negative (pRSV JunD) expression vector or pRSV empty vector as a control; C, 2 g of TRAF2 dominant negative (TRAF2 DN) expression vector or pRK5 empty vector as a control; D, 2 g of TRAF6 dominant negative (TRAF6 DN) expression vector or pcDNA3 empty vector as a control; and E, 2 g of CBF1 dominant negative (pJH282) expression vector or pSG5 empty vector as a control. The transfected cultures were split into two flasks. 24 h after transfection, one flask was incubated with 30 g/ml anti-CD40 mAb 3/23 for 24 h while the remaining flask was left untreated. Luciferase activity was determined 48 h after transfection. Luciferase activities were normalized to pRLTK activity and expressed as the mean S.E. of three independent transfection experiments. Samples treated with 3/23 are depicted in solid black boxes, whereas untreated samples are in white. Statistical analysis was performed by Student’s t test. * and *** denote p 0.05 and p 0.005, respectively.

Article Snippet: Mouse anti-human CD40 monoclonal antibody LOB7.6 was obtained from Serotec, UK.

Techniques: Transfection, Dominant Negative Mutation, Expressing, Plasmid Preparation, Control, Incubation, Luciferase, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity

doi: 10.1074/jbc.M117.812784

Figure Lengend Snippet: Characterization of MxA G domain variants. A, antiviral activity of the G domain variants in a FLUAV minireplicon system. 293T cells were co-transfected with expression plasmids for the MxA variants (300 ng) and the minireplicon system of VN/04, including a reporter construct encoding firefly luciferase under the control of the viral promoter. After 24 h, firefly luciferase activity was determined and normalized to the activity of constitutively co-expressed Renilla luciferase. Results are presented relative to the activity in the absence of MxA, the vector control (see “Experimental procedures” for calculation), and as arithmetic means ± S.D. of three independent experiments. Protein expression of FLAG-tagged MxA, viral NP, and actin was determined by Western blot analysis. B, restriction of FLUAV replication by G domain variants in tissue culture. A549 cells were transfected with MxA expression plasmids (500 ng) and 24 h later infected with SC35MNS1_2A_GFP-NEP (H7N7) at an MOI of 0.5. After fixation of the cells at 10 h post-infection, MxA was stained, and cells were analyzed by FACS. MxA-positive cells were selected, and the percentage of infected (GFP-positive) cells was determined. The percentage of GFP-positive cells expressing the inactive mutant T103A was set to 100%. Arithmetic means ± S.D. (error bars) of four independent experiments are shown. C, G domain dimer of GMPPCP-bound (gray) stalkless MxA (4P4S, G domain A in yellow (residues 70–340), G domain B in blue (residues 68–340)) (8). Positions of MxA G domain variations are highlighted in red. Amino acid residues of WT MxA are shown in stick representations. D, analytical gel-filtration analysis of the indicated mutants in the presence and absence (apo) of GDP-AlFx. E, nucleotide binding analysis of monomeric MxA G domain variants by ITC at 8 °C. GTPγS was titrated stepwise into the protein solution. The resulting heat changes were integrated, and the obtained values were fitted to a quadratic binding equation (one-site binding model). The following KD values were derived from the fittings: M527D (black), KD = 13 ± 5 μm, n = 0.83 ± 0.08; M527D/N220D (red), KD = 13 ± 2 μm, n = 0.50 ± 0.04; M527D/G255E (blue), KD = 5 ± 2 μm, n = 0.48 ± 0.18; M527D/V268M (green), KD = 17 ± 3 μm, n = 0.54 ± 0.04. The MxA constructs showed a varying degree of precipitation in these assays, which may explain the reduced binding numbers. F, protein concentration-dependent GTPase activities of monomeric M527D (□) and M527D/N220D (○) were determined at 37 °C by an HPLC-based assay. The mean kobs was calculated from two independent experiments for each concentration. The error bars show the range of the two data points. mAU, milliabsorbance units.

Article Snippet: The following primary antibodies were used: anti-FLAG (clone M2, mouse monoclonal, F3165, Sigma-Aldrich), anti-HA (rabbit polyclonal, H6908, Sigma-Aldrich), anti-FLUAV NP (clone AA5H, mouse monoclonal, MCA400, Bio-Rad), anti-THOV NP (rabbit polyclonal ( 5 )), anti-β-actin (rabbit polyclonal, ab8227, Abcam), and anti-MxA (mouse monoclonal, M143 ( 44 )).

Techniques: Activity Assay, Transfection, Expressing, Construct, Luciferase, Plasmid Preparation, Western Blot, Infection, Staining, Mutagenesis, Filtration, Binding Assay, Derivative Assay, Protein Concentration, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity

doi: 10.1074/jbc.M117.812784

Figure Lengend Snippet: MxA G-interface variants have no dominant-negative effect on WT MxA. A, co-immunoprecipitation of WT MxA with G domain variants. 293T cells were co-transfected with HA-tagged WT MxA and FLAG-tagged MxA mutants. At 24 h post-transfection, cell lysates were subjected to FLAG-specific immunoprecipitations (IP). Precipitates and whole-cell lysates (WCL) were analyzed by Western blotting. B, effect of MxA G domain variants on the antiviral activity of WT MxA in the FLUAV minireplicon system of VN/04, as described in the legend to Fig. 2A. HA-tagged WT MxA (300 ng) was co-transfected with the components of the minireplicon and increasing amounts (50, 100, and 200 ng) of the indicated FLAG-tagged MxA variants. Protein expression was monitored by Western blot analysis. Data are presented as described in the legend to Fig. 2A. C, GTPase activity of M527D can be stimulated by the monomeric G domain mutant N220D. M527D (2.5 μm) was incubated with increasing concentrations of M527D/N220D, and GTPase activity was measured as described in the legend to Fig. 2F. Vertical lines in the Western blots indicate cuts combining two blots of one experiment run in parallel. Error bars, S.D.

Article Snippet: The following primary antibodies were used: anti-FLAG (clone M2, mouse monoclonal, F3165, Sigma-Aldrich), anti-HA (rabbit polyclonal, H6908, Sigma-Aldrich), anti-FLUAV NP (clone AA5H, mouse monoclonal, MCA400, Bio-Rad), anti-THOV NP (rabbit polyclonal ( 5 )), anti-β-actin (rabbit polyclonal, ab8227, Abcam), and anti-MxA (mouse monoclonal, M143 ( 44 )).

Techniques: Dominant Negative Mutation, Immunoprecipitation, Transfection, Western Blot, Activity Assay, Expressing, Mutagenesis, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity

doi: 10.1074/jbc.M117.812784

Figure Lengend Snippet: Antiviral activity of MxA stalk variants. A, antiviral activity against FLUAV in the VN/04 minireplicon system, as described in the legend to Fig. 2A. The vector control and the antiviral activity of WT MxA and T103A have already been shown in Fig. 2A. B, antiviral activity against THOV in the SiAr126 minireplicon system. MxA variants (100 ng of expression plasmids) and the components of the minireplicon system, including a reporter construct encoding firefly luciferase under the control of the THOV promoter and Renilla luciferase to monitor transfection efficiency, were co-expressed in 293T cells. Firefly luciferase activity was measured in the cell lysates at 24 h post-transfection and normalized to the activity of the Renilla luciferase. Data are presented as described in the legend to Fig. 2A. C, restriction of FLUAV replication by stalk variants in A549 cells analyzed by FACS, as described in the legend to Fig. 2B. The antiviral activities of WT MxA and T103A have already been shown in Fig. 2B. D and E, antiviral activity against VSV. D, 293T cells were co-transfected with FLAG-tagged MxA variants (300 ng) and VSV-G (300 ng). At 24 h post-transfection, cells were infected with VSV*ΔG(Luc) at an MOI of 1. Another 24 h later, supernatants were collected, the cells were harvested, and firefly luciferase activity was measured (VLP infection). E, the supernatants containing newly produced VLPs were used to infect naive 293T cells, which were lysed 24 h later to determine firefly luciferase activity (VLP titration). The values are presented relative to the activity in the absence of MxA, the vector control (see “Experimental procedures” for calculation). Arithmetic means ± S.D. (error bars) of three biological replicates are shown. F, restriction of VSV replication by stalk variants in tissue culture. 24 h after transfection with MxA expression plasmids (500 ng), A549 cells were infected with VSV-GFP at an MOI of 0.5 for 6.5 h. Fixed cells stained with an MxA-specific antibody were analyzed by FACS. MxA-positive cells were selected, and the percentage of infected (GFP-positive) cells was determined. The percentage of GFP-positive cells expressing the inactive mutant T103A was set to 100%. Results are displayed as arithmetic means ± S.D. of three independent experiments. Protein expression of FLAG-tagged MxA, actin, FLUAV, and THOV NP was verified by Western blot analyses. Vertical lines in the Western blots indicate cuts combining two blots of one experiment run in parallel. The color code of the bars is explained under “Results.”

Article Snippet: The following primary antibodies were used: anti-FLAG (clone M2, mouse monoclonal, F3165, Sigma-Aldrich), anti-HA (rabbit polyclonal, H6908, Sigma-Aldrich), anti-FLUAV NP (clone AA5H, mouse monoclonal, MCA400, Bio-Rad), anti-THOV NP (rabbit polyclonal ( 5 )), anti-β-actin (rabbit polyclonal, ab8227, Abcam), and anti-MxA (mouse monoclonal, M143 ( 44 )).

Techniques: Activity Assay, Plasmid Preparation, Expressing, Construct, Luciferase, Transfection, Infection, Produced, Titration, Staining, Mutagenesis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity

doi: 10.1074/jbc.M117.812784

Figure Lengend Snippet: Characterization of the MxA stalk variants V470G and E516del. A, antiviral activity against RVFV. MxA (100 ng) and RVFV-N and -L (50 ng each) were co-expressed in 293T cells. 24 h post-transfection, cells were infected with Rift Valley fever VLPs encoding firefly luciferase as a reporter. Luciferase activities were measured 24 h later and are presented relative to the activity in the absence of MxA, the vector control (see “Experimental procedures” for calculation). Arithmetic means ± S.D. (error bars) of four biological replicates are shown. Western blot analysis was performed to control protein expression of FLAG-tagged MxA and actin. Significance was calculated using one-way analysis of variance with Dunnett's post hoc test. ns, not significant; **, p ≤ 0.01; ****, p ≤ 0.0001. B, protein concentration–dependent GTPase activities of WT MxA (gray; same control as in Fig. 5A) and V470G (green), as in Fig. 5A. C, analytical gel filtration of WT MxA, M527D (gray and black; same controls as in Fig. 5B), and V470G (green) in the absence of nucleotide, as described in the legend to Fig. 2D. D, co-immunoprecipitation of HA-tagged WT MxA (500 ng) with the FLAG-tagged variants (IP) V470G and E516del (500 ng) as described in the legend to Fig. 5C. Precipitates and whole-cell lysates (WCL) were analyzed by Western blotting. E, nuclear co-translocation of the MxA stalk variants V470G and E516del with WT MxA. Artificial nuclear forms of WT and MxA variants carrying an HA tag and the NLS of the SV40 large T antigen (HA-NLS-MxA) were co-expressed with FLAG-tagged WT MxA or MxA variants in HeLa cells. At 24 h post-transfection, cells were fixed and stained with antibodies directed against the HA tag (red) and the FLAG tag (green). The right column displays the overlay of the two signals. F, effect of different amino acid substitutions at position 470 on the antiviral activity of MxA. The antiviral activity of the mutants against FLUAV was determined in the VN/04 minireplicon system, as described in the legend to Fig. 2A.

Article Snippet: The following primary antibodies were used: anti-FLAG (clone M2, mouse monoclonal, F3165, Sigma-Aldrich), anti-HA (rabbit polyclonal, H6908, Sigma-Aldrich), anti-FLUAV NP (clone AA5H, mouse monoclonal, MCA400, Bio-Rad), anti-THOV NP (rabbit polyclonal ( 5 )), anti-β-actin (rabbit polyclonal, ab8227, Abcam), and anti-MxA (mouse monoclonal, M143 ( 44 )).

Techniques: Activity Assay, Transfection, Infection, Luciferase, Plasmid Preparation, Western Blot, Expressing, Protein Concentration, Filtration, Immunoprecipitation, Translocation Assay, Staining, FLAG-tag

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 1. Expression of TRPC6 protein in human glioma tissue. A) Representative immunoblots of total lysates extracted from human glioma (six samples shown) and normal brain (six samples shown) tissues probed with anti–transient re ceptor potential canonical (TRPC)6 (106 kDa) or anti-TRPC3 (97 kDa) antibodies. GAPDH served as the protein loading control. B and C) Quantification of TRPC6 and TRPC3 protein levels in glioma (n = 33) and normal brain (n = 17) tissues from the immunoblots. Each dot repre sents the band density of TRPC6 and 3 proteins normalized to that of the corre sponding GAPDH in each sample. n = number of samples. Means and 95% confidence intervals are represented by open lines and error bars. **P < .001 vs normal, calculated using the two-sided Student t test. D) Representative immu nohistological staining samples of human glioma or normal brain tissues with anti- TRPC6 antibody. a–i) Three glioblastoma multiforme (GBM, grade 4) samples; j–l) anaplastic oligodendrocytoma (grade 3 oligodendrocytoma); m–o) grade 2 astro cytoma; and p–r) normal (para-tumor) tissues. Sections were stained by the 3,3′-diaminobenzidine (DAB) staining method to detect TRPC6 protein in the tissues and were counterstained with hematoxylin to stain the nucleus. Serial sections of the same samples were used for hematoxylin and eosin (HE) staining. Magnified ×40 panels represented the white rectangles in the ×10 panels. Scale bar = 100 µm. E) Bar graph representa tion of TRPC6 immunohistological scores in glioma of different grades. The per centage of sections with different scores (strong, moderate, and weak) in each grade is shown. Grade 4 glioma (GBM), n = 18; grade 3, n = 7; grades 1 and 2 combined, n = 8. n = number of samples. F) Expression of TRPC6 mRNA in glioma and normal tissues detected by in situ hybridization. Immunostaining by anti- TRPC6 antibody (DAB, ×40) on serial sections of the same samples used for immunohistological staining. Sense probe (×40) was used as a negative con trol, and the antisense probe (×40) was used to detect the TRPC6 mRNA. Scale bar = 100 µm.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Expressing, Western Blot, Control, Staining, In Situ Hybridization, Immunostaining

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 2. Functional activity of transient receptor potential canonical 6 (TRPC6) channels in glioma cells. Fura-2 AM imaging showing induc tion and attenuation of transient elevation of [Ca2+]i in U87 cells. R refers to the ratio of the emission at 500 nm induced by 340 and 380 nm exci tation, captured at 6-second intervals. DR/R was calculated as (R 2 Rbaseline)/Rbaseline, Rbaseline being the mean value of R during pretreatment. The recording time in minutes is shown. Squares represent the mean values. Error bars = 95% confidence intervals. Corresponding bar charts show the area of DR/R curve in the Fura-2 AM imaging, 3 minutes after addition of platelet-derived growth factor-BB (PDGF-BB) or 10 minutes after addition of cyclopiazonic acid (CPA). In all bar charts, the y-axis shows the absolute value of the area divided by 100. Means and upper 95% confidence intervals are shown. All P values were calculated using the two-sided Student t test. Ctrl = control; n = number of cells. A) Induction with 50 ng/mL PDGF-BB (Ctrl, solid square) and attenuation with 20 µM SKF96365 (SKF, open square). Medium was supplemented with calcium (Ca2+). B) Bar chart corresponds to (A). Ctrl, n = 48; SKF, n = 32. *P = .014 vs Ctrl. Data from three independent experiments are shown in (A) and (B). C) Induction with PDGF-BB in cells infected with adenovirus-expressing wild-type TRPC6 (WTC6, solid square) and at tenuation in cells expressing the dominant-negative form of TRPC6 (DNC6, open square). Duration of infection was 48 hours. Medium was supplemented with Ca2+. D) Bar chart corresponds to (C). Cells infected with adenovirus expressing green fluorescent protein (GFP) served as the control. GFP, n = 48; DNC6, n = 51; WTC6, n = 45. **P = .001, GFP vs DNC6, and **P < .001, WTC6 vs DNC6. Data from at least three indepen dent experiments are shown in (C) and (D). E) Induction with PDGF-BB in cells infected with GFP (solid square) or DNC6 (open square) in Ca2+- free medium (0 Ca2+) and Ca2+-containing medium (2 mM Ca2+). Smaller peaks represent the internal Ca2+ release induced by PDGF-BB, and larger peaks represent the extracellular Ca2+ entry on addition of 2 mM

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Functional Assay, Activity Assay, Imaging, Derivative Assay, Control, Infection, Expressing, Dominant Negative Mutation

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 3. Effect of inhibition of transient re ceptor potential canonical 6 (TRPC6) activity or expression on glioma cell growth. A and B) Number of U251 and U87 cells infected with adenovirus expressing green fluorescent protein (GFP), DNC6, or WTC6. Cell numbers were normalized to those at day 0. Means and upper 95% confidence intervals from three independent experiments performed in dupli cate are shown. *P < .05, **P < .01, GFP or WTC6 vs DNC6. C) Representative images of U251 and U87 colonies infected with adeno virus expressing GFP, DNC6, or WTC6. Colonies stained with crystal violet after 14 days of seed ing are shown. D) Quantification of U251 and U87 colonies (shown in C) by measuring the optical density at 595 nm (OD595). OD595 values of DNC6 and WTC6 groups were normalized to that of GFP group. Means and upper 95% confi dence intervals are shown. Data are representa tive of four independent experiments, performed in duplicate. *P < .05, **P < .01, GFP or WTC6 vs DNC6. E) U251 colonies infected with lentivi rus-based scrambled short hairpin RNA (shRNA) (Ctrl), shTRPC6-1 (C6-1), and shTRPC6-2 (C6-2). Colonies stained with crystal violet after 14 days of seeding were shown. Ctrl = control. F) Differential interference contrast (DIC) and fluo rescent (GFP) images of typical single colonies formed by U251 cells infected with the indi cated lentiviral constructs—scrambled shRNA (Ctrl), shTRPC6-1 (C6-1), and shTRPC6-2 (C6-2). The duration of infection was 72 hours before seeding. The lentivirus was GFP tagged. Ctrl = control. Images were taken 14 days after seed ing. Scale bar = 100 µm. Merge panels show the merged DIC and GFP images. G) Quantification of the U251 colonies (shown in E) by measuring the OD595. Means and upper 95% confidence intervals are shown. Data are representative of four independent experiments performed in duplicate. *P = .032, **P < .001 vs Ctrl. All P values were calculated using the two-sided Student t test.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Inhibition, Activity Assay, Expressing, Infection, Virus, Staining, shRNA, Control, Construct

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 4. Effect of inhibition of transient receptor potential canonical 6 (TRPC6) activity or expres sion on G2 phase arrest. A and B) Flow cytometric analysis of U251 DNA content after infection with adenovirus expressing green fluorescent protein (GFP), DNC6, and WTC6 for 72 hours or the indi cated lentivirus-based short hairpin RNA (shRNA) constructs (Ctrl, C6-1, and C6-2), 5–10 days after infection. DNA content is shown as 2n and 4n in the x-axis. 2n = cells in G0/G1 phase, and 4n = cells in the G2/M phase. Data are representative of four independent experiments. Ctrl = control, scrambled shRNA (Ctrl); C6-1 = shTRPC6-1 and C6-2 = shTRPC6-2. C) Quantitative real-time poly merase chain reaction and immunoblot analysis (inset) showing knockdown of CACNA1G in U251 cells by lentivirus-based shRNA constructs against the CACNA1G (CACNA1G RNA interference [RNAi]). Control (Ctrl) RNAi was nonsense shRNA. Data are representative of five independent exper iments. TUBA1A served as the protein loading control. Error bar = upper 95% confidence inter val. **P < .001 vs Ctrl RNAi. D) Flow cytometric analysis of U251 cell cycle after infection with the lentivirus-based shRNA construct (CACNA1G RNAi) (shown in C), 5–10 days after infection. Data shown are a representative of six independent experiments. E) Mitotic index of U251 cells after infection with adenovirus expressing GFP, DNC6, and WTC6 for 72 hours. Mitotic index (%) is the percentage of mitotic cells in total cells. Means and upper 95% confidence interval from three independent experiments are shown. **P = .005, GFP vs DNC6, and *P = .022, WTC6 vs DNC6. F) Effect of TRPC6 knockdown on the level of phos phorylated cyclin-dependent kinase 1 at tyrosine15 (P-CDK1 [Y15]). Immunoblot analysis of total U251 cell lysates using the indicated antibodies. Proteins were extracted approximately 10 days after infection. C6-1 and -2 lanes show approxi mately 40% and 60% decrease in TRPC6 level and increased in P-CDK1 (Y15) level by two- or four fold, respectively. TRPC3 blots show that TRPC6 knockdown did not have an effect on its homolog. TUBA1A served as the protein loading control. Data are representative of three independent ex periments. All P values were calculated using the two-sided Student t test.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Inhibition, Activity Assay, Infection, Expressing, shRNA, Construct, Control, Western Blot, Knockdown

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 6. Effect of inhibition of transient re ceptor potential canonical 6 (TRPC6) activity on radiosensitization of glioma cells. A) Representative crystal violet staining of the colonies formed by U251 cells 11 days after irradiation with 0, 2, 4, 6, and 8 Gy. Cells were treated with vehicle (Ctrl) or 10 µM SKF96365 (SKF) 24 hours before irradiation. Ctrl = con trol. B and C) Surviving fraction of U251 cells after irradiation with 0, 2, 4, 6, and 8 Gy. Cells were treated with vehicle (Ctrl) or 10 µM SKF96365 (SKF) for 24 hours (B), or infected with adenovirus-based green fluorescent protein (GFP) or DNC6 at multiplicity of infec tion = 2, for 72 hours (C) before irradiation. Ctrl = control. Data were representative of five independent experiments, performed in dupli cate. Error bars = 95% confidence interval. **P = .006, SKF vs Ctrl at 2 Gy; **P = .005, SKF vs Ctrl at 4 Gy; **P < .001, GFP vs DNC6 at 2 Gy; **P = .002, GFP vs DNC6 at 4 Gy. All P values were calculated using the two-sided Student t test.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Inhibition, Activity Assay, Staining, Irradiation, Infection, Control

Journal: Journal of the National Cancer Institute

Article Title: Essential role of TRPC6 channels in G2/M phase transition and development of human glioma.

doi: 10.1093/jnci/djq217

Figure Lengend Snippet: Figure 7. Effect of inhibition of transient re ceptor potential canonical 6 (TRPC6) activity on the development of xenografted human glioma. A) The tumor volumes in green fluorescent protein (GFP)-, DNC6-, and WTC6-infected groups (n = 7 per group) were determined every 5 days for 30 days after implantation of U87 cells. Means and upper 95% confidence inter vals were shown. *P < .05, **P < .01, GFP or WTC6 vs DNC6. P values were calculated using the two-sided Student t test. B) Representative photographs of nude mice bearing xenografted tumors on day 30 of implantation. C) Scatter diagram of individual tumor volume of xeno grafted tumors on day 30 of implantation. Each symbol represents a single tumor. Error bars represent the interquartile range. *P = .014, GFP vs DNC6, **P < .001 WTC6 vs DNC6. P values were calculated using the two-sided Student t test. D) Survival curve was plotted by Kaplan– Meier analysis. Mice bearing glioma from DNC6-infected U87 cells survived longer than GFP- or WTC6-infected U87 cells. The number of mice (n) in each group is indicated. Each curve was compared using the two-sided log- rank test. P < .001, DNC6 vs GFP or WTC6. Number of mice at risk at each time point is also shown. E) Hematoxylin and eosin staining of representative coronal sections of the mouse brains 20 days after implantation. The tumors were derived from GFP-, DNC6-, and WTC6- infected U87 cells, respectively.

Article Snippet: The probe sequence targeting 443–1436 bases of the human TRPC6 mRNA (GenBank accession NM_004621) was amplified by polymerase chain reaction (PCR) (DNA Engine Peltier Thermal Cycler; Bio-Rad Laboratories, Hercules, CA) using human TRPC6 cDNA (OriGene Technologies, Rockville, MD) as the template.

Techniques: Inhibition, Activity Assay, Infection, Staining, Derivative Assay

Journal: eLife

Article Title: A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity

doi: 10.7554/eLife.05449

Figure Lengend Snippet: All data plots: average and S.E.M.; ‘*’: p < 0.05, ‘**’: p < 0.01, ‘***’: p < 0.001 by the two-tailed Mann–Whitney U test. The number on top of each bar indicates the biological replicate number. ( A ) Transfection of Gpr52 siRNAs (Gpr52_si1∼3) in the mouse striatal cells (STHdh Q7/Q111 ) lowers Htt levels, as detected by Htt antibodies MW1, 2166, ab1 and 2B7. MW1 is the polyQ antibody that detects only the mHtt protein, whereas 2166, 2B7 and ab1 detects both mHtt and wtHtt. Left panels: representative western-blots; Hdh5 is the Htt siRNA used as the positive control for Htt knock-down. Neg is the non-targeting siRNA used as the negative control. Right panel: western-blot quantification from multiple replicates. ( B ) Infection of lentiviruses expressing Gpr52 shRNAs (Gpr52_sh1∼2) lowers Htt in primary striatal but not cortical neurons cultured from Hdh Q140/Q140 knock-in mice. Left panels: representative western-blots. Right panel: western-blot quantification for the normalized 3B5H10 signals from multiple replicates. ( C ) Heterozygous knockout of Gpr52 lowers Htt in vivo in the striata but not cortices of Hdh Q140/Q7 knock-in mice in vivo. The mice were obtained by crossing the heterozygous Gpr52 knockout mice with the Hdh Q140/Q140 knock-in mice. Littermates between 40 to 69 days of age were analyzed. Left panels: representative western-blots. Right panel: western-blot quantification of the normalized MW1 signals from multiple mouse samples. Each dot represents the signal from a single mouse. ( D ) Left panels: Immunostaining of HD patient iPS-derived striatal-like neurons. Differentiated neurons from HD patient's iPS cells express molecular markers for striatal medium spiny neurons: Tuj1, GABA and DARPP32. Scale bar: 50 μM. Right panels: Transfection of human Gpr52 siRNAs (hGpr52_si1∼2) in the HD patient iPS-derived neurons lowers Htt levels detected by both western-blots and HTRF. HTT3 is the Htt siRNA used as the positive control for Htt knock-down. Bar plot represents the normalized mHtt levels detected by HTRF using the 2B7/MW1 antibody pair. DOI: http://dx.doi.org/10.7554/eLife.05449.003

Article Snippet: Coverslip cultures were fixed in 4% paraformaldehyde for 15–20 min, washed with 1× PBS 10 min three times and incubated in a blocking buffer (10% donkey serum and 0.2% triton X-100 in PBS) for 60 min and then incubated with primary antibodies: Tuj1 (Covance, Princeton, NJ, USA, cat. no. 14971502, 1:5000), GABA (Sigma, cat. no. A0310, 1:200), DARPP32 (Millipore, cat. no. AB1656, 1:1000), Htt antibodies 2B7 ( ) (1:200), 2050 or 2051 (Bio-rad, MCA2050 or MCA2051, 1:200), Rab22A antibody (abcam, cat. no. ab137093), or Rab39B antibody (abcam, cat. no. ab154826) overnight at 4°C.

Techniques: Two Tailed Test, MANN-WHITNEY, Transfection, Western Blot, Positive Control, Knockdown, Negative Control, Infection, Expressing, Cell Culture, Knock-In, Knock-Out, In Vivo, Immunostaining, Derivative Assay

Journal: eLife

Article Title: A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity

doi: 10.7554/eLife.05449

Figure Lengend Snippet: All experiments are performed in the mouse striatal cell line STHdh Q7/Q111 , and all data are plotted as average and S.E.M. ‘*’: p < 0.05, ‘**’: p < 0.01, ‘***’: p < 0.001. The number on top of each bar indicates the biological replicate number. ( A ) Changes of cAMP levels measured by the cAMP-Glo assay (Promega). Gpr52 siRNAs were tranfected for 3 days, whereas the compound treatment (forskolin: 1 µM; reserpine: 10 µM) lasts for 24 hr; statistical analyses performed by the two-tailed Mann–Whitney U test. ( B ) Htt level measured by the 2B7/2166 HTRF upon treatment of different doses of the Gpr52 agonist reserpine for 48 hr, with transfection of Gpr52 siRNA (Gpr52_si1) vs the non-targeting control (Neg_si), n = 4. ( C ) Htt level measured by the 2B7/2166 HTRF when transfected with hGPR52 cDNA titrated with the empty control vector at different percentages (X-axis), n = 6; statistical analysis performed by the one-way ANOVA and post-hoc Dunnett's test. ( D ) Left and middle : Representative western-blots of STHdh Q7/Q111 cells transfected with the Gpr52 siRNA (Gpr52_si2) and then treated with the indicated PKA modulators or cAMP analogs (forskolin and cAMP analogs: 1 µM; H89: 50 µM). Calnexin has been used as a loading control. The Bars plot : Htt level changes (%) measured by 2B7/2166 HTRF of the total lysates with the same treatments as in the western-blot samples. ( E ) Confocal microscopy experiments showing that HTT proteins are enriched in the perinuclear and co-localize with the endoplasmic reticulum (ER) marker calreticulin upon treatment of Rp-cAMP or 8-pCpt-2′-O-Me-cAMP (8-pCpt-cAMP for short). Upper panels : representative images showed the immunofluorescent signals of Htt (green), ER marker (red, only in the third and fourth columns) and DAPI (blue) in STHdh Q7/Q111 cells treated by vehicle, 1 μM Rp-cAMP or 1 μM 8-cpt-cAMP. Scale bars , 20 μM. The two panels on the right side are magnified images from the left for visualizing the co-localization. Yellow pixels indicate co-localization. Lower left plot : the percentage of cells showing clear perinuclear pattern in each samples. The pattern was judged blindly. Lower middle and right plots : co-localization parameters including Pearson's coefficient and overlap coefficient (mean and S.E.M.). Numbers in indicate the number of cells analyzed for each treatment from five or more biological replicates. DOI: http://dx.doi.org/10.7554/eLife.05449.006

Article Snippet: Coverslip cultures were fixed in 4% paraformaldehyde for 15–20 min, washed with 1× PBS 10 min three times and incubated in a blocking buffer (10% donkey serum and 0.2% triton X-100 in PBS) for 60 min and then incubated with primary antibodies: Tuj1 (Covance, Princeton, NJ, USA, cat. no. 14971502, 1:5000), GABA (Sigma, cat. no. A0310, 1:200), DARPP32 (Millipore, cat. no. AB1656, 1:1000), Htt antibodies 2B7 ( ) (1:200), 2050 or 2051 (Bio-rad, MCA2050 or MCA2051, 1:200), Rab22A antibody (abcam, cat. no. ab137093), or Rab39B antibody (abcam, cat. no. ab154826) overnight at 4°C.

Techniques: Glo Assay, Two Tailed Test, MANN-WHITNEY, Transfection, Control, Plasmid Preparation, Western Blot, Confocal Microscopy, Marker

Journal: eLife

Article Title: A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity

doi: 10.7554/eLife.05449

Figure Lengend Snippet: Left: Representative western-blots of STHdh Q7/Q111 cells transfected with the Gpr52 siRNA (Gpr52_si2) versus control siRNA, and then with constitutively active or dominant negative Rap1 (Rap1CA or Rap1DN) or Rap2 (Rap2CA or Rap2DN). The Bars plot: Htt level reduction (%) measured by 2B7/2166 HTRF of the total lysates of cells with same transfections as in the western-blots. DOI: http://dx.doi.org/10.7554/eLife.05449.009

Article Snippet: Coverslip cultures were fixed in 4% paraformaldehyde for 15–20 min, washed with 1× PBS 10 min three times and incubated in a blocking buffer (10% donkey serum and 0.2% triton X-100 in PBS) for 60 min and then incubated with primary antibodies: Tuj1 (Covance, Princeton, NJ, USA, cat. no. 14971502, 1:5000), GABA (Sigma, cat. no. A0310, 1:200), DARPP32 (Millipore, cat. no. AB1656, 1:1000), Htt antibodies 2B7 ( ) (1:200), 2050 or 2051 (Bio-rad, MCA2050 or MCA2051, 1:200), Rab22A antibody (abcam, cat. no. ab137093), or Rab39B antibody (abcam, cat. no. ab154826) overnight at 4°C.

Techniques: Western Blot, Transfection, Control, Dominant Negative Mutation

Journal: eLife

Article Title: A striatal-enriched intronic GPCR modulates huntingtin levels and toxicity

doi: 10.7554/eLife.05449

Figure Lengend Snippet: ( A ) Genomic loci of Rabgap1l and Gpr52. ( B ) qPCR quantification of the Rabgap1l mRNA level of STHdh cells transfected with Rabgap1l siRNAs or the non-targeting control siRNA (Neg). 50–80% knock-down could be achieved by siRNA transfection in these cells. ( C ) Western-blot ( left ) and HTRF ( right ) experiments showing that Rabgap1l knock-down by siRNA increases the Htt level in the STHdh cells. Hdh5 and B01 are Htt siRNAs used as positive controls. For HTRF, the 2B7/2166 antibody pair was used. Data are plotted as mean and S.E.M, n = 16 for non-targeting siRNA control (Neg) samples, and n = 12 for Rabgap1l siRNA transfected samples. ‘***’: P < 0.001 by the two-tailed Mann–Whitney U-test. DOI: http://dx.doi.org/10.7554/eLife.05449.011

Article Snippet: Coverslip cultures were fixed in 4% paraformaldehyde for 15–20 min, washed with 1× PBS 10 min three times and incubated in a blocking buffer (10% donkey serum and 0.2% triton X-100 in PBS) for 60 min and then incubated with primary antibodies: Tuj1 (Covance, Princeton, NJ, USA, cat. no. 14971502, 1:5000), GABA (Sigma, cat. no. A0310, 1:200), DARPP32 (Millipore, cat. no. AB1656, 1:1000), Htt antibodies 2B7 ( ) (1:200), 2050 or 2051 (Bio-rad, MCA2050 or MCA2051, 1:200), Rab22A antibody (abcam, cat. no. ab137093), or Rab39B antibody (abcam, cat. no. ab154826) overnight at 4°C.

Techniques: Transfection, Control, Knockdown, Western Blot, Two Tailed Test, MANN-WHITNEY