Journal: PLoS ONE

Article Title: Mice lacking EFA6C/Psd2, a guanine nucleotide exchange factor for Arf6, exhibit lower Purkinje cell synaptic density but normal cerebellar motor functions

doi: 10.1371/journal.pone.0216960

Figure Lengend Snippet: Primary antibodies used in this study.

Article Snippet: One day after transfection, cells were harvested with a solution consisting of 125 mM TrisHCl (pH 6.8), 4% SDS, 20% glycerol and 10% 2-mercaptoethanol, and boiled for 5 min. Total lysates of cerebellar samples (10 μg) and HeLa cells over-expressing FLAG-mEFA6C were subjected to immunoblotting with antibodies against EFA6A [ ], EFA6C [ ] (Cat. No. 17404-1-AP, ProteinTech, Rosemont, IL), EFA6D [ ], BRAG1 [ ], BRAG2 [ ], BRAG3 [ ], cytohesin-2 [ ], Arf6 [ ], calbindin (Cat. No. Calbindin-Rb-Se-1, RRID: AB_2571568, Frontier Institute, Ishikari, Japan) and mGluR1a (Cat. No. mGluR1a-Rb-Af811, PRID:AB_2571799, Frontier Institute) as listed in .

Techniques: Recombinant, Purification

Journal: PLoS ONE

Article Title: Mice lacking EFA6C/Psd2, a guanine nucleotide exchange factor for Arf6, exhibit lower Purkinje cell synaptic density but normal cerebellar motor functions

doi: 10.1371/journal.pone.0216960

Figure Lengend Snippet: (A) Schematic illustration of the strategy to generate EFA6C KO mice. The gRNA sequence targeting exon 1 of the mouse EFA6C gene and the PAM sequence are shown in bold and red, respectively. (B) Sequence alignment of exon 1 in wild-type and mutant alleles. Note the deletion of 112 nucleotides in the mutant allele. The sequences of the target and PAM are shown in bold and red, respectively. (C) PCR genotyping using primers indicated in Fig 1B. The expected sizes of wild-type and mutant amplicons were 320 and 208 bp, respectively. The positions and sizes (bp) of DNA markers are indicated on the left. (D, E) Immunoblot analyses. The lysates of cerebella from three male mice of each genotype and HeLa cells exogenously expressing FLAG-EFA6C were subjected to immunoblotting with antibodies against EFA6C N-terminal region (EFA6C[N]), EFA6C C-terminal region (EFA6C[C]), EFA6A, EFA6D, BRAG1–3, cytohesin-2 (Cyth2), Arf6, calbindin and mGluR1. The same blots were reprobed with anti-α-tubulin IgG. Note that immunoreactive bands for EFA6C were completely absent in the cerebellar lysates of homozygous mice, and there were no apparent differences in the protein expression of Arf6 GEFs, Arf6, calbindin and mGluR1 in the cerebellar lysates between the two genotypes. The positions and sizes (kDa) of molecular weight markers are indicated on the right. (F) Quantification of the protein expression of Arf6 GEFs, Arf6, calbindin and mGluR1. Relative optical densities for immunoreactive bands normalized to those for α-tubulin are shown as mean ± SEM of relative ratio to the wild-type control group set as 1.

Article Snippet: One day after transfection, cells were harvested with a solution consisting of 125 mM TrisHCl (pH 6.8), 4% SDS, 20% glycerol and 10% 2-mercaptoethanol, and boiled for 5 min. Total lysates of cerebellar samples (10 μg) and HeLa cells over-expressing FLAG-mEFA6C were subjected to immunoblotting with antibodies against EFA6A [ ], EFA6C [ ] (Cat. No. 17404-1-AP, ProteinTech, Rosemont, IL), EFA6D [ ], BRAG1 [ ], BRAG2 [ ], BRAG3 [ ], cytohesin-2 [ ], Arf6 [ ], calbindin (Cat. No. Calbindin-Rb-Se-1, RRID: AB_2571568, Frontier Institute, Ishikari, Japan) and mGluR1a (Cat. No. mGluR1a-Rb-Af811, PRID:AB_2571799, Frontier Institute) as listed in .

Techniques: Sequencing, Mutagenesis, Western Blot, Expressing, Molecular Weight, Control

Journal: Biology of the cell

Article Title: Actin Binding to the BAR Domain and Arf GAP Activity of ASAP1 Coordinately Control Actin Stress Fibers and Focal Adhesions.

doi: 10.1111/boc.70005

Figure Lengend Snippet: FIGURE 3 Effect of knockdown of endogenous ASAP1 on focal adhesions (FAs) and stress fibers (SFs) is rescued by expression of ASAP1 from an ectopic promoter. U2OS cells, with cDNA expression cassettes for ASAP1 under control of a tetracycline promoter integrated into the chromosome using a lentivirus vector, were plated in 35 mm cell culture plates and treated with either nontargeting (control Si) or siRNA targeting the 3′UTR of ASAP1 (ASAP1 siRNA) as indicated. After 24 h, doxycycline (0.5 µg/mL) was added as indicated. Two days later, cells were harvested and replated on fibronectin coated cover slips for 6 h in serum free medium. The cells were then fixed and stained for actin and paxillin. (A) Immunoblot of cell lysates for ASAP1 under the indicated conditions. The top panel is a blot using an antibody to ASAP1, the middle panel uses an antibody to the epitope tag fused to the ectopically expressed ASAP1. Note there is some expression without doxycycline when endogenous ASAP1 is knockdown and expression with doxycycline is greater with reduced endogenous ASAP1. This observation is reproducible and is the subject of another project. β-Actin was used as a loading control. (B) Quantification of FAs. Conditions are indicated in the figure. (C) Quantification of bundled actin. Bundled actin was quantified using the imageJ plugin RidgeDetector (left panel) and by determining the number of peaks in nine line profiles as described in Figure 2 and “Section 2” (right panel). Results using either method for quantification were similar. (D) Representative images. SFs are indicated with arrows and FAs with arrowheads. Results were analyzed with one-way (Panel C) and two-way (Panel B) analysis of variance (ANOVA). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Mouse (Ms) anti-HA (6E2), Ms anti-paxillin (Clone 349), Ms anti-GAPDH (14C10), Ms anti-α-Actinin, and Rb anti-Arf6 (D12G6), Rb anti-HA (C29F4), Rb anti-Myosin IIa (E7Y9O), and Rb anti-β-Actin Abs from Cell Signaling.

Techniques: Knockdown, Expressing, Control, Plasmid Preparation, Cell Culture, Staining, Western Blot

Journal: Biology of the cell

Article Title: Actin Binding to the BAR Domain and Arf GAP Activity of ASAP1 Coordinately Control Actin Stress Fibers and Focal Adhesions.

doi: 10.1111/boc.70005

Figure Lengend Snippet: FIGURE 4 Reduced Arf5 expression affects stress fibers (SFs) and focal adhesions (FAs). U2OS cells were treated with either a pool of siRNA or a single siRNA targeting Arf5. After 3 days, cells were harvested. Aliquots were lysed and immunoblotted. Cells were plated on fibronectin coated coverslips for 5–6 h, fixed, stained for actin and paxillin, and imaged. (A) Immunoblot of lysates of U2OS cells treated with the indicated siRNA for the reduction of Arf5 expression. Specificity of knockdown was checked by blotting for Arf6. β-Actin was used as a loading control. (B) Quantification of the Immunoblot. Blots were quantified using fluorescent secondary antibodies detected using an Odyssey Imager (LiCOR). (C) Effect of Arf5 knockdown on FAs in U2OS. FAs were quantified as described in Figure 2 and in “Section 2.” (D) Effect of Arf5 knockdown on cellular distribution of FAs. The distance of FAs (>1 µm2) from the cell edge was determined as described in “Section 2.” The density of paxillin containing plaques at relative distances from the edge of the cell is plotted, 0 being the edge of the cell. Thirty-eight cells for the mock condition, 40 cells for the control siRNA condition and 31 cells each for the Arf5 targeted siRNA condition were analyzed. (E) Effect of Arf5 knockdown on SFs. Bundled actin was quantified as described in Figure 2 and in “Section 2.” (F) Representative images. Cells were treated with nontargeting (control) or Arf5 targeted (#17 supplied by Dharmacon) siRNA. For Panel (C), summary of three experiments is presented. Each experiment was normalized to the mock transfection control. The data was analyzed using two-way analysis of variance (ANOVA) with a repeated measures design. Panels (B) and (E) were analyzed using one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Mouse (Ms) anti-HA (6E2), Ms anti-paxillin (Clone 349), Ms anti-GAPDH (14C10), Ms anti-α-Actinin, and Rb anti-Arf6 (D12G6), Rb anti-HA (C29F4), Rb anti-Myosin IIa (E7Y9O), and Rb anti-β-Actin Abs from Cell Signaling.

Techniques: Expressing, Staining, Western Blot, Knockdown, Control, Transfection

Journal: Biology of the cell

Article Title: Actin Binding to the BAR Domain and Arf GAP Activity of ASAP1 Coordinately Control Actin Stress Fibers and Focal Adhesions.

doi: 10.1111/boc.70005

Figure Lengend Snippet: FIGURE 5 Both GTP binding and hydrolysis by Arf5 are required for the maintenance of focal adhesions (FAs) and stress fibers (SFs). (A) Representative images of U2OS cells expressing the indicated mutant of Arf5. Cells were transfected with plasmids for expression of the indicated Arf5 mutant. Control cells were treated with transfection reagent and an empty expression vector. After 24 h, the cells were replated on fibronectin coated coverslips for 6 h, fixed and stained for actin, paxillin, or the ectopic Arf5 (identified by the HA tag fused to Arf5). Arrowheads indicate paxillin containing plaques at the edge of the cell. (B) Distribution of FAs in U2OS cells expressing Arf5 mutants. The density of paxillin containing plaques at relative distance from the edge of the cell is plotted, 0 being the edge of the cell. 36, 36, 36, and 38 cells were analyzed for control, Arf5-HA, [T31N]Arf5-HA, and [I46D]Arf5-HA. (C) Effect of expressing Arf5 and mutants of Arf5 on number and size of FAs in U2OS cells. The number and size of paxillin-containing plaques in U2OS cells expressing the indicated Arf5 mutants were determined as described in (Chen and Kroog, 2010) and in “Section 2.” (D) Effect of expression Arf5 and Arf5 mutants on bundles of actin between 5 and 10 µm in length. Bundled actin was quantified using ridge detector in ImageJ. (E) Effect of Arf mutants on bundled actin determined by quantifying peaks in line profiles. The number of peaks in nine line profiles in two ROIs/cell was determined as described in “Section 2.” The mean values from three experiments, each analyzing 10–20 cells/condition, are presented. (F) Localization of [I46D]Arf5-HA and ASAP1. Representative images are shown. Examples of apparent colocalization of [I46D]Arf5 with ASAP1 at cell edge are indicated with arrows. (G) Colocalization of Arf and Arf mutants with ASAP1. Cells expressing the indicated recombinant Arf were stained for the epitope tag on Arf (HA) and ASAP1. Colocalization was assessed by determining Pearson’s coefficients. (H) Immunoblot of cell lysates. Antibodies specific for Arf5 and for the HA epitope were used to detect Arf5. Note the epitope tagged protein has a fusion of a 6 Histidine tandem followed by the HA epitope, runs more slowly than endogenous Arf5. β-Actin was used as a loading control. FA data were analyzed by two-way analysis of variance (ANOVA). Other experiments were analyzed by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Mouse (Ms) anti-HA (6E2), Ms anti-paxillin (Clone 349), Ms anti-GAPDH (14C10), Ms anti-α-Actinin, and Rb anti-Arf6 (D12G6), Rb anti-HA (C29F4), Rb anti-Myosin IIa (E7Y9O), and Rb anti-β-Actin Abs from Cell Signaling.

Techniques: Binding Assay, Expressing, Mutagenesis, Transfection, Control, Plasmid Preparation, Staining, Recombinant, Western Blot

Journal: Biology of the cell

Article Title: Actin Binding to the BAR Domain and Arf GAP Activity of ASAP1 Coordinately Control Actin Stress Fibers and Focal Adhesions.

doi: 10.1111/boc.70005

Figure Lengend Snippet: FIGURE 6 Effect of ASAP1 on actin cytoskeleton and focal adhesions (FAs) requires GAP activity and the BAR domain. Lentivirus was used to integrate cDNA with the open reading frame for the indicated mutants of ASAP1 ([R485K]ASAP1-HA or [ΔBAR]ASAP1-HA) under control of a tet-on promoter. Each cell line was treated with either nontargeting (control) or siRNA targeting the 3′UTR of ASAP1 (ASAP1 SiRNA) for a day, and then treated with either doxycycline (0.5 µg/mL) or vehicle for an addition 2 days before plating on fibronectin coated covers for 6 h, fixing, staining for actin and paxillin, and imaging. (A) Representative images. Examples of stress fibers (SFs) are indicated by arrows, FAs by arrowheads. (B) Effect of reduced ASAP1 and expression of [R485K]ASAP1-HA on SFs and FAs. (i) Bundled actin, quantified by determining the number of peaks from line profiles, was taken as a measure of SFs. The summary of two experiments, analyzing 10–20 cells/condition/experiment, is shown. (ii) FAs. Summary data from two experiments for [R485K]ASAP1 and three experiments for [ΔBAR]ASAP1 is presented. (iii) Protein levels determined by immunoblotting. Total ASAP1 (includes endogenous and the mutants) was detected with an antibody to ASAP1. The doxycycline-induced protein was detected with an antibody to the HA epitope tag. β-actin was used as a loading control. (C) Effect of reduced endogenous ASAP1 and expression of [ΔBAR]ASAP1-HA on SFs and FAs. Subpanels (i–iii) parallel those in Part B.

Article Snippet: Mouse (Ms) anti-HA (6E2), Ms anti-paxillin (Clone 349), Ms anti-GAPDH (14C10), Ms anti-α-Actinin, and Rb anti-Arf6 (D12G6), Rb anti-HA (C29F4), Rb anti-Myosin IIa (E7Y9O), and Rb anti-β-Actin Abs from Cell Signaling.

Techniques: Activity Assay, Control, Staining, Imaging, Expressing, Western Blot