Journal:

Article Title: ?-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates ?-adrenoceptor switching from G s to G i

doi: 10.1073/pnas.262787199

Figure Lengend Snippet: Isoprenaline-induced phosphorylation of the β2AR in HEK 293 cells. In all these experiments at the indicated times, HEK 293 cells overexpressing the β2AR were harvested, and the PKA phosphorylation status of the anti-FLAG-immunopurified β2AR was assessed by using the PKA substrate antibody. In all instances lanes were loaded equally with immunopurified receptor. (a) Cells were challenged with either isoprenaline (Iso, 10 μM) alone or together with rolipram (Iso + roli, 10 μM). Lanes were loaded equally with immunopurified receptor. (b) Quantification of the data in a for n = 3 experiments with means ± SD. (c) Cells were treated for 8 min with isoprenaline (10 μM) plus rolipram (10 μM) in either the absence or presence of the PKA inhibitor H89 (1 μM). (d) Cells were treated for 8 min with isoprenaline (10 μM) plus rolipram (10 μM) either without or with transfection of protein kinase inhibitor (PKI), the PKA inhibitor (21). (e) As indicated, cells were transfected to express D556A-PDE4D5 before being treated for 8 min with isoprenaline (10 μM) before analysis. (f) Quantification of the data shown in d for n = 3 experiments with means ± SD.

Article Snippet: A phospho-serine PKA substrate antibody (Cell Signaling Technologies, Beverly, MA) was used to detect PKA phosphorylation of the β 2 AR.

Techniques: Transfection

Journal:

Article Title: ?-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates ?-adrenoceptor switching from G s to G i

doi: 10.1073/pnas.262787199

Figure Lengend Snippet: A schematic representation of the role of arrestin-recruited PDE4 in regulating the “switching” of the β2AR from Gs to Gi stimulation. Agonist occupancy of the β2AR initially leads to coupling to Gs, which causes activation of adenylyl cyclase, elevated cAMP levels, and activation of PKA, which is able to phosphorylate the β2AR. Concomitantly, agonist occupancy also leads to GRK-mediated phosphorylation of the β2AR, which allows for the recruitment of β-arrestin together with bound PDE4. PKA phosphorylation of the β2AR confers switching from Gs to Gi, with consequent activation of ERK1/2. However, β-arrestin-recruited PDE4 provides a negative feedback loop, the role of which is to attenuate local cAMP levels and thus the ability of membrane PKA to phosphorylate the β2AR. This action of β-arrestin-recruited PDE4 is uncovered by dominant negative PDE4, which replaces the active endogenous recruited PDE4 to ablate the negative feedback loop and thus accentuate switching to Gi.

Article Snippet: A phospho-serine PKA substrate antibody (Cell Signaling Technologies, Beverly, MA) was used to detect PKA phosphorylation of the β 2 AR.

Techniques: Activation Assay, Dominant Negative Mutation

Journal: Human Molecular Genetics

Article Title: PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity

doi: 10.1093/hmg/dds302

Figure Lengend Snippet: Genes whose expression was statistically associated with TPMT activity ( P -value lower than 5 × 10 −5 ) in 87 Human HapMap CEU cell lines

Article Snippet: PACSIN2 and TPMT mRNA were quantified by real-time PCR using the commercial TaqMan Gene Expression Assay (Hs00200589_m1 and Hs00909010_g1, respectively) from Applied Biosystems.

Techniques: Expressing, Activity Assay, Sequencing

Journal: Human Molecular Genetics

Article Title: PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity

doi: 10.1093/hmg/dds302

Figure Lengend Snippet: Multivariate logistic regression model for the effects of PACSIN2, SLCO1B1 and TPMT genotypes on the incidence of GI toxicity

Article Snippet: PACSIN2 and TPMT mRNA were quantified by real-time PCR using the commercial TaqMan Gene Expression Assay (Hs00200589_m1 and Hs00909010_g1, respectively) from Applied Biosystems.

Techniques:

Journal: Human Molecular Genetics

Article Title: PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity

doi: 10.1093/hmg/dds302

Figure Lengend Snippet: Genes whose mRNA was differentially expressed after knock-down of PACSIN2 in human NALM6 leukemia cells

Article Snippet: PACSIN2 and TPMT mRNA were quantified by real-time PCR using the commercial TaqMan Gene Expression Assay (Hs00200589_m1 and Hs00909010_g1, respectively) from Applied Biosystems.

Techniques: Knockdown, Binding Assay, Dominant Negative Mutation

Journal: Human Molecular Genetics

Article Title: PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity

doi: 10.1093/hmg/dds302

Figure Lengend Snippet: Enrichment analysis of mRNA differentially expressed in human NALM6 leukemia cells, between PACSIN2 controls and knock-down

Article Snippet: PACSIN2 and TPMT mRNA were quantified by real-time PCR using the commercial TaqMan Gene Expression Assay (Hs00200589_m1 and Hs00909010_g1, respectively) from Applied Biosystems.

Techniques: Membrane

Journal: Human Molecular Genetics

Article Title: PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity

doi: 10.1093/hmg/dds302

Figure Lengend Snippet: Classification and regression tree analysis for genetic predictors influencing those patients at an increased risk for GI toxicity. The three SNPs have independent effects, with TPMT being ranked before SLCO1B1 and PACSIN2.

Article Snippet: PACSIN2 and TPMT mRNA were quantified by real-time PCR using the commercial TaqMan Gene Expression Assay (Hs00200589_m1 and Hs00909010_g1, respectively) from Applied Biosystems.

Techniques:

Journal: Human Molecular Genetics

Article Title: PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity

doi: 10.1093/hmg/dds302

Figure Lengend Snippet: The frequency of severe (Grade 3–4) GI toxicity during consolidation therapy in the validation cohort, the Italian AIEOP-BFM-ALL 2000 and genotypes in PACSIN2; PACSIN2 SNP rs2413739 and rs737782 resulted associated with the incidence of GI toxicity in this cohort (P-values from logistic regression model, 0.032 and 0.015, respectively).

Article Snippet: PACSIN2 and TPMT mRNA were quantified by real-time PCR using the commercial TaqMan Gene Expression Assay (Hs00200589_m1 and Hs00909010_g1, respectively) from Applied Biosystems.

Techniques: Biomarker Discovery

Journal: Human Molecular Genetics

Article Title: PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity

doi: 10.1093/hmg/dds302

Figure Lengend Snippet: TPMT activity in NALM6 cells heterologously expressing TPMT*1 or TPMT*3A and with knock-down of PACSIN2. After knock-down of PACSIN2, a significant (30%) decrease in TPMT activity was observed in cells heterologously expressing TPMT*1 (P-value Welch t-test = 0.0036).

Article Snippet: PACSIN2 and TPMT mRNA were quantified by real-time PCR using the commercial TaqMan Gene Expression Assay (Hs00200589_m1 and Hs00909010_g1, respectively) from Applied Biosystems.

Techniques: Activity Assay, Expressing, Knockdown

Journal: Human Molecular Genetics

Article Title: PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity

doi: 10.1093/hmg/dds302

Figure Lengend Snippet: Diagram describing the analysis strategy to identify the most important trans-genes whose expression and SNPs are associated with TPMT activity in the HapMap CEU population. Among the genes whose expression is highly associated with TPMT activity (false discovery rate 1.6%), PACSIN2 is the only one that has significant SNPs (P-value <0.0005) associated with TPMT activity.

Article Snippet: PACSIN2 and TPMT mRNA were quantified by real-time PCR using the commercial TaqMan Gene Expression Assay (Hs00200589_m1 and Hs00909010_g1, respectively) from Applied Biosystems.

Techniques: Expressing, Activity Assay

Journal: Human Molecular Genetics

Article Title: PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity

doi: 10.1093/hmg/dds302

Figure Lengend Snippet: The box plot shows TPMT activity in 286 patients with ALL as a function of the TPMT rs1142345 [A719G]/PACSIN2 rs2413739 multilocus genotype. There were no GG genotypes for TPMT rs1142345 and therefore all variants genotypes for TPMT are heterozygous. SNPs in both genes had a significant association with TPMT enzyme activity in a univariate analysis (P-values from linear model with formula TPMT activity–genotype, considering an additive effect for the genotype: TPMT = 6.0 × 10−16; PACSIN2 P = 0.027). Moreover, these SNPs (TPMT, PACSIN2) were also significantly related to TPMT activity in a multivariate analysis that combined the two SNPs in a multilocus genotype (TPMT P-value from linear model = 3.0 × 10−16 and PACSIN2 P-value from linear model = 0.011). The PACSIN2 genotype had a significant effect even in the subgroup with the wild-type TPMT genotype (P-value from linear model = 0.031) and in the subgroup with the variant TPMT genotype (P-value from linear model = 0.0091).

Article Snippet: PACSIN2 and TPMT mRNA were quantified by real-time PCR using the commercial TaqMan Gene Expression Assay (Hs00200589_m1 and Hs00909010_g1, respectively) from Applied Biosystems.

Techniques: Activity Assay, Variant Assay

Journal: Human Molecular Genetics

Article Title: PACSIN2 polymorphism influences TPMT activity and mercaptopurine-related gastrointestinal toxicity

doi: 10.1093/hmg/dds302

Figure Lengend Snippet: The frequency of severe (Grade 3–4) GI toxicity during consolidation therapy in patients with ALL treated according to the St Jude Total 13B protocol as a function of TPMT rs1142345, PACSIN2 rs2413739, SLCO1B1 rs11045879 genotypes. Panel A shows barplots reporting the percentage of patients developing GI toxicity and the corresponding genotypes. Panel B represents cumulative incidence plots of GI toxicity and the corresponding genotypes for the same patients. There were no GG genotypes for TPMT rs1142345 and therefore all variants genotypes for TPMT are heterozygous. Among nine patients with a variant TPMT allele, the frequency of gastrointestinal toxicity was 33%, compared with 7.2% in patients with the wild-type TPMT (P-value from logistic regression model = 0.0058); PACSIN2 SNP rs2413739 also had a significant association with GI toxicity during consolidation therapy: the frequency of toxicity was 2.1, 9.1 and 13.2%, respectively, for the CC, CT and TT allele (P-value from logistic regression model = 0.046). An SLCO1B1 SNP (rs11045879) that we have previously reported to be associated with GI toxicity in ALL patients (9) was also associated with the incidence of GI toxicity: none of the patients with the SLCO1B1 CC or CT genotype had GI toxicity, whereas 11.8% of the patients with the wild-type SLCO1B1 TT genotype had GI toxicity (P-value from logistic regression model = 0.012).

Article Snippet: PACSIN2 and TPMT mRNA were quantified by real-time PCR using the commercial TaqMan Gene Expression Assay (Hs00200589_m1 and Hs00909010_g1, respectively) from Applied Biosystems.

Techniques: Variant Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1 1 2

doi:

Figure Lengend Snippet: BART decreases the activity of Rac1 resulting in inhibition of PDAC cell motility and invasion. (A) The amount of active, GTP-loaded Rac1 in S2-013 and PANC-1 cells that were mock transfected (Neo-1) or transfected with control, scrambled (Scr-1), or BART (siBART-1 and 2) siRNA was determined using a GST-PAK-CRIB pull-down assay. Precipitates were examined by Western blot analysis using an anti-Rac1 antibody. Data are representative of three independent experiments. (B) Two S2-013 clones transfected with siRNA for BART were pretreated with or without the Rac1 inhibitor (NSC23766), and the amount of active GTP-loaded Rac1 and Cdc42 and RhoA was analyzed with GST pull-down assays using GST-PAK-CRIB and GST-Rhotekin, respectively. Precipitates were examined by Western blot analysis using anti-Rac1, anti-Cdc42, and anti-RhoA antibodies. Total levels of Rac1, Cdc42, and RhoA protein were used to normalize the data. Data are representative of three independent experiments. (C) Confluent control and BART RNAi S2-013 cells treated as in B were wounded. The number of cells that migrated into an initially cell-free scratch was counted. Cells in four defined areas per group per experiment were quantified. Data are representative of three independent experiments. Bars, SD; columns, mean. *P < .005, **P < .001 compared with nontreated cells. (D) Control and BART RNAi S2-013 cells treated as in B were plated on Matrigel invasion chambers. Invaded cells in four fields per group were counted. Data are representative of three independent experiments. Bars, SD; columns, mean. *P < .005, **P < .001 compared with nontreated cells.

Article Snippet: GST-tagged Rac1 protein (8 µg) attached to glutathione-agarose beads (Cytoskeleton) was incubated either with GTPγS or with GDP at a final concentration of 1 mM in 20 µl of reaction buffer (50 mM HEPES [pH 7.4], 100 mM NaCl, 10 mM MgCl 2 , 5 mM EDTA, 1 mM DTT) at 30°C for 15 minutes.

Techniques: Activity Assay, Inhibition, Transfection, Control, Pull Down Assay, Western Blot, Clone Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1 1 2

doi:

Figure Lengend Snippet: BART binds to Rac1 at the leading edges of migrating cells. (A) Immunoprecipitation of endogenous BART or Rac1 from S2-013 cells. Anti-BART or anti-Rac1 immunoprecipitates were examined by Western blot analysis using anti-BART and anti-Rac1 antibodies. Rabbit IgG and mouse IgG monoclonal antibodies were used as isotype controls for anti-BART and anti-Rac1 antibodies, respectively. Data are representative of three independent experiments. (B) Immunocytochemical staining of S2-013 cells using anti-BART (green) and anti-Rac1 (red) antibodies. Arrow indicates colocalized BART and Rac1 in lamellipodial-like protrusions; blue, DAPI staining. Bar, 10 µm. (C) Confluent S2-013 cells were wounded. After 4 hours, the cells were immunostained using anti-BART (green) and anti-Rac1 (red) antibodies. Arrows indicate colocalized BART and Rac1 at the leading edge; blue, DAPI staining. Bar, 10 µm.

Article Snippet: GST-tagged Rac1 protein (8 µg) attached to glutathione-agarose beads (Cytoskeleton) was incubated either with GTPγS or with GDP at a final concentration of 1 mM in 20 µl of reaction buffer (50 mM HEPES [pH 7.4], 100 mM NaCl, 10 mM MgCl 2 , 5 mM EDTA, 1 mM DTT) at 30°C for 15 minutes.

Techniques: Immunoprecipitation, Western Blot, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1 1 2

doi:

Figure Lengend Snippet: BART binds to an active form of Rac1. (A) GST-tagged Rac1 was incubated with GDP or GTPγS and was then used in pull-down experiments with the recombinant BART protein. Precipitates were examined by Western blot analysis using anti-BART and anti-Rac1 antibodies. Data are representative of three independent experiments. (B) GST-tagged Rac1, a dominant-negative mutated Rac1 form, Rac1N17, or PAK-CRIB was incubated with S2-013 cell lysates, followed by GST-pull-down assays. Precipitates were examined by Western blot analysis using anti-BART and anti-Rac1 antibodies. Data are representative of three independent experiments. (C) Densitometric analysis of the results of B. The level of BART in the precipitates was assessed after normalizing BART signals to Rac1 signals. (D) S2-013 cells were pretreated with or without the Rac1 inhibitor (NSC23766) and were immunocytochemically stained using anti-BART (green) and anti-Rac1 (red) antibodies. Arrows indicate colocalized BART and Rac1 in lamellipodial-like protrusions; blue, DAPI staining. Bars, 10 µm.

Article Snippet: GST-tagged Rac1 protein (8 µg) attached to glutathione-agarose beads (Cytoskeleton) was incubated either with GTPγS or with GDP at a final concentration of 1 mM in 20 µl of reaction buffer (50 mM HEPES [pH 7.4], 100 mM NaCl, 10 mM MgCl 2 , 5 mM EDTA, 1 mM DTT) at 30°C for 15 minutes.

Techniques: Incubation, Recombinant, Western Blot, Dominant Negative Mutation, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1 1 2

doi:

Figure Lengend Snippet: BART has GAP activity toward Rac1. (A) The BART-rescue construct was transfected into control and BART knockdown cells of S2-013, and 48 hours later, the amount of active GTP-loaded Rac1 was determined by GST-pull-down assays using GST-PAK-CRIB. Precipitates were examined by Western blot analysis using an anti-Rac1 antibody. Data are representative of three independent experiments. Closed arrowhead indicates exogenous BART; open arrowhead, endogenous BART. (B) The possibility that BART might have a GAP function for Rac1 GTPase was assayed by in vitro GAP assays using human Rac1 with or without the human p50 RhoGAP protein together with recombinant BART protein. GST was used as a negative control for RhoGAP and BART protein. GAP activity was assayed by measurement of the phosphate generated by hydrolysis of GTP. Data are representative of three independent experiments and are shown as means ± SEM. *P < .005 compared with their respective controls.

Article Snippet: GST-tagged Rac1 protein (8 µg) attached to glutathione-agarose beads (Cytoskeleton) was incubated either with GTPγS or with GDP at a final concentration of 1 mM in 20 µl of reaction buffer (50 mM HEPES [pH 7.4], 100 mM NaCl, 10 mM MgCl 2 , 5 mM EDTA, 1 mM DTT) at 30°C for 15 minutes.

Techniques: Activity Assay, Construct, Transfection, Control, Knockdown, Western Blot, In Vitro, Recombinant, Negative Control, Generated

Journal: Neoplasia (New York, N.Y.)

Article Title: BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1 1 2

doi:

Figure Lengend Snippet: BART inhibits the translocation of Rac1 to the plasma membrane. (A) The mock control vector or myc-tagged BART-rescue construct was transfected into BART knockdown cells of S2-013, and 48 hours later, the cells were incubated on fibronectin for 1 hour. GST-pull-down assays were performed using GST-PAK-CRIB. The precipitates were immunoblotted with anti-myc and anti-Rac1 antibodies. Data are representative of three independent experiments. (B) Control (Neo-1 and Scr-1) and BART RNAi (siBART-1 and 2) S2-013 cells treated as in A were fractionated, and particulate/membranous (p) and soluble/cytosolic (s) fractions were analyzed by Western blot using anti-Rac1 antibody. Asterisk indicates fibronectin-stimulated Rac1 in the particulate fraction of BART RNAi cells. Data are representative of three independent experiments. (C) Scrambled control (Scr-1) and BART RNAi (siBART-1) S2-013 cells treated as in A were immunocytochemically stained using anti-Rac1 (green) and anti-myc (red) antibodies. Blue indicates DAPI staining. The corresponding differential interference contrast (DIC) images are shown. Bars, 10 µm.

Article Snippet: GST-tagged Rac1 protein (8 µg) attached to glutathione-agarose beads (Cytoskeleton) was incubated either with GTPγS or with GDP at a final concentration of 1 mM in 20 µl of reaction buffer (50 mM HEPES [pH 7.4], 100 mM NaCl, 10 mM MgCl 2 , 5 mM EDTA, 1 mM DTT) at 30°C for 15 minutes.

Techniques: Translocation Assay, Membrane, Control, Plasmid Preparation, Construct, Transfection, Knockdown, Incubation, Western Blot, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: BART Inhibits Pancreatic Cancer Cell Invasion by Rac1 Inactivation through Direct Binding to Active Rac1 1 2

doi:

Figure Lengend Snippet: BART inhibits membrane lamellipodial formation by inhibiting peripheral actin-cytoskeletal rearrangements. (A)Scrambled control (Scr-1) and BART knockdown (siBART-1) S2-013 cells were incubated on fibronectin for 1 hour, and then actin was immunocytochemically stained using phalloidin (red) (left panels). Blue indicates DAPI staining. The corresponding DIC images are shown. Bars, 10 µm. Western blot with anti-BART antibody showing the siBART-1 cells compared with the control Scr-1 cells incubated on fibronectin (right panels). (B) Quantification of data shown in A; the values represent the number of scrambled control and BART knockdown S2-013 cells with lamellipodial protrusion. Cells in four fields per group were counted. Data are representative of three independent experiments. Bars, SD; columns, mean. *P < .005 compared with control cells. (C) BART RNAi S2-013 cells (siBART-1) were pretreated with or without the Rac1 inhibitor (NSC23766) and then incubated on fibronectin for 1 hour. The cells were immunocytochemically stained using anti-Rac1 antibody (green), and cellular actin was then stained using phalloidin (red). Blue indicates DAPI staining. Bars, 10 µm. (D) The mock control vector or BART-rescue construct was transfected into BART knockdown S2-013 cells (siBART-1), and 48 hours later, the cells were incubated on fibronectin for 1 hour. The cells were immunocytochemically stained using anti-myc antibody (green), and cellular actin was then stained using phalloidin (red). Blueindicates DAPI staining. The corresponding DIC images are shown. Bars, 10 µm.

Article Snippet: GST-tagged Rac1 protein (8 µg) attached to glutathione-agarose beads (Cytoskeleton) was incubated either with GTPγS or with GDP at a final concentration of 1 mM in 20 µl of reaction buffer (50 mM HEPES [pH 7.4], 100 mM NaCl, 10 mM MgCl 2 , 5 mM EDTA, 1 mM DTT) at 30°C for 15 minutes.

Techniques: Membrane, Control, Knockdown, Incubation, Staining, Western Blot, Plasmid Preparation, Construct, Transfection