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-Rac1 Binding Assay In Vitro 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, 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-Rac1 Binding Assay In Vitro 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-Rac1 Binding Assay In Vitro 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-Rac1 Binding Assay In Vitro 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, 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-Rac1 Binding Assay In Vitro 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, Plasmid Preparation, Construct, Transfection, 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-Rac1 Binding Assay In Vitro 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, Staining, Western Blot, Plasmid Preparation, Construct, Transfection