arp3 proteins  (Cytoskeleton Inc)

Journal: eLife

Article Title: ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization

doi: 10.7554/eLife.85167

Figure Lengend Snippet: ( A ) Schematic overview of CDC42-WASP-stimulated ARP2/3-dependent actin polymerization based on the cited literature. The process involves ARP2/3 complex, WASP (VCA) as nucleation promoting factor, filamentous actin (F-actin), and monomeric actin (G-actin). In the initial step, CDC42 is activated by GEF-catalyzed exchange of GDP to GTP. Active CDC42 (CDC42-GTP) binds to the GTP-binding domain (GBD) on WASP, thereby displacing the VCA domain. While the V-verpolin-like motif binds actin monomer (G-actin), C-central and A-acidic domains bind and activate the ARP2/3 complex. Conformational changes induced by the binding of the ARP2/3 complex promote its binding to the actin filament, which is strengthened by the additional interaction of the ARP2/3 complex with WASP (VCA)-G-actin. Further conformational changes will secure the ARP2/3 complex on the filament and allow its binding to the actin monomer and the polymerization of the newly nucleated filament. Actin polymerizes at the fast-growing/barbed end, elongating toward the plasma membrane and the ARP2/3 complex would cross-link newly polymerizing filament to the existing filament. ( B ) ERK3 co-precipitates with active RAC1 and CDC42 in complex with ARP2/3. Active RAC1/CDC42 pull-down was performed using control and ERK3 knockdown human mammary epithelial cells (HMECs). Levels of the active RAC1 and CDC42 were assessed as well as the co-immunoprecipitation levels of ERK3, ARP2, ARP3, and ARPC1A. Levels of the total protein expression were evaluated in the total cell lysates (TCL) and Ponceau S staining was used as a loading control. ( C–F ) ERK3 regulates F-actin levels in vitro and in vivo. ( C ) Western blot analyses of control (CRISPR Co) and ERK3 -depleted (CRISPR ERK3 ) HMECs are presented alongside with representative confocal images of F-actin staining. ( D, E ) In vivo analysis of F- and G-actin levels in HMECs upon ERK3 knockdown. ( D ) Representative western blot analyses of the enriched F- and G-actin fractions as well as the ERK3 knockdown validation and total actin levels in the TCL are presented. ( E ) F- and G-actin levels were quantified, and ratios were calculated from five (n = 5) independent experiments and are presented as mean ± SEM; *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001, unpaired t -test. Analyses of ERK3-dependent regulation of F-actin levels in cancerous MDA-MB231 cells is presented in . Cellular colocalization between endogenous ERK3 and the ARP2/3 was assessed in the absence of CDC42 and is presented in . ( F ) Effect of full-length ERK3 on ARP2/3-dependent pyrene actin polymerization was assessed using a pyrene actin polymerization assay. Polymerization induced by the VCA domain of WASP that served as a positive control (green) as well as the ARP2/3 (orange) and ERK3 protein alone (blue) are shown for reference. Actin alone (black) was used to establish a baseline of polymerization. Fluorescence at 360/415 was measured over time and is presented as mean fold change from at least three independent experiments after normalization to the first time point within the respective group. ARP2/3-dependent actin polymerization was measured in the presence of both ERK3 and WASP (VCA) domain, and the results are depicted in . Figure 5—source data 1. Full membrane scans for western blot images for and . Figure 5—source data 2. Prism and Excel file for . Figure 5—source data 3. Prism and Excel file for .

Article Snippet: Purified RAC1, CDC42, and ARP3 proteins or ARP2/3 protein complex (Cytoskeleton) were used as bait and full-length GST-ERK3 (SignalChem) was used as a titrant protein.

Techniques: Binding Assay, Immunoprecipitation, Expressing, Staining, In Vitro, In Vivo, Western Blot, CRISPR, Polymerization Assay, Positive Control, Fluorescence

Journal: eLife

Article Title: ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization

doi: 10.7554/eLife.85167

Figure Lengend Snippet: ( A ) Coomassie-stained 10% SDS-PAGE gel with 1 mg of the ARP2/3 protein complex (Cytoskeleton) presenting all the subunits. ( B ) Binding of increasing concentrations of recombinant GST-ERK3 to the ARP2/3 complex was measured by ELISA as described in the 'Materials and methods' section. ( C ) The interaction between GST-ERK3 and ARP3 was measured in vitro using GST-pull-down assay as described in the 'Materials and methods' section. ( D ) Binding affinity of the recombinant GST-ERK3 protein and ARP3 was assessed by ELISA as described in the 'Materials and methods' section and mean absorbance (Abs) ± SEM from three independent experiments is presented. ( E ) Co-immunoprecipitation (IP) of ARP2/3 protein complex and ERK3 was performed in HMECs using ARP3 antibody. Levels of precipitated ARP3 as well as co-IP of ARP2 and ERK3 were assessed. IgG control was included to determine the specificity of the interaction. Total cell lysate (TCL) was included to present expression levels of the verified interacting partners. Ponceau S staining was used as a loading control. ( F, G ) Actin phenotype of the human mammary epithelial cells (HMECs) was validated upon stable overexpression of the ARP3 non-phosphorylatable (S418A) and the phospho-mimicking (S418D) mutant, respectively. Wild type (WT) ARP3 was used as a control for the mutants and empty vector (EV) served negative control for the overexpression itself. ( F ) F-actin expression and organization in the negative (S418A) and phospho-mimicking (S418D) ARP3 mutant was visualized by green phalloidin and merged with the Hoechst staining of the nuclei. Four representative confocal images are presented. Images of EV-transfected and ARP3 WT-overexpressing HMECs are presented as controls. ( G ) Western blot validation of the overexpression efficiency and phosphorylation of ARP3 at S418. Anti-V5-tag antibody was used to detect levels of exogenous ARP3 WT, S418A, and S418D. Expression levels of the endogenous ARP3 were assessed as well as the phosphorylation at S418, total actin was validated. Ponceau S staining was used as a loading control. ( H ) Detection of the S418 phosphorylation of ARP3 in CRISPR ERK3 HMECs presented in . ( I, J ) Effect of the ARP3 mutant overexpression on F-actin levels was quantified using F/G actin in vivo assay. ( I ) Representative western blot analyses of F- and G-actin levels detected in fractions obtained from EV, ARP3 WT, S418A, S418D HMECs. ( J ) Quantification of the F/G actin ratios was performed for three (n = 3) independent experiments and is presented as mean ± SEM; *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001, one-way ANOVA, Tukey’s post-test. ( K–M ) Effect of ERK3 depletion on dense F-actin phenotype of the ARP3 S418D-overexpressing HMECs. HMECs stably overexpressing ARP3 S418D were transduced with lentiviral particles targeting ERK3 (shERK3) and stable knockdown was established as described in the 'Materials and methods' section. Cells were further subjected to analyses of the F-actin levels. ( K ) IF staining with Oregon Green Phalloidin 488 to visualize F-actin levels and organization. Scale bars 28 µm. ( L, M ) Effect of the ERK3 knockdown on F-actin levels was quantified in the ARP3 S418D mutant overexpressing HMECs using F/G actin in vivo assay. ( L ) Representative western blot analyses of F/G actin levels. ARP3 S418D-(V5-tagged) overexpression and ERK3 knockdown efficiency were validated in TCL. Actin and Ponceau S staining were used as loading controls. ( M ) Calculated ratios of F/G actin are presented as mean ± SEM from three (n = 3) independent experiments; *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001, paired t -test. Colocalization of endogenous ERK3 with endogenous and exogenous ARP3 mutant (S418D) was verified, and further effect of the ERK3 depletion on the RAC1 and CDC42 activity was assessed in ARP3 S418D-overexpressing HMECs and presented in . Figure 6—source data 1. Full membrane scans for western blot images for . Figure 6—source data 2. Prism and Excel file for . Figure 6—source data 3. Prism and Excel file for . Figure 6—source data 4. Prism and Excel file for . Figure 6—source data 5. Prism and Excel file for .

Article Snippet: Purified RAC1, CDC42, and ARP3 proteins or ARP2/3 protein complex (Cytoskeleton) were used as bait and full-length GST-ERK3 (SignalChem) was used as a titrant protein.

Techniques: Staining, SDS Page, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, In Vitro, Pull Down Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Over Expression, Mutagenesis, Plasmid Preparation, Negative Control, Transfection, Western Blot, CRISPR, In Vivo, Stable Transfection, Transduction, Activity Assay

Journal: eLife

Article Title: ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization

doi: 10.7554/eLife.85167

Figure Lengend Snippet: ERK3 directly binds and activates the ARP2/3 protein complex as well as the CDC42 and RAC1 Rho GTPases. Activation of the ARP2/3 complex and RAC1/CDC42 is required for nucleation of the new actin filaments, elongation, and branching into the lamellipodia and filopodia. ERK3 regulates actin-rich protrusions, which play a direct role in cell motility.

Article Snippet: Purified RAC1, CDC42, and ARP3 proteins or ARP2/3 protein complex (Cytoskeleton) were used as bait and full-length GST-ERK3 (SignalChem) was used as a titrant protein.

Techniques: Activation Assay

Journal: eLife

Article Title: ERK3/MAPK6 dictates CDC42/RAC1 activity and ARP2/3-dependent actin polymerization

doi: 10.7554/eLife.85167

Figure Lengend Snippet:

Article Snippet: Purified RAC1, CDC42, and ARP3 proteins or ARP2/3 protein complex (Cytoskeleton) were used as bait and full-length GST-ERK3 (SignalChem) was used as a titrant protein.

Techniques: Sequencing, shRNA, CRISPR, Recombinant, Mutagenesis, Plasmid Preparation, Cell Fractionation, In Vivo, Transduction, Concentration Assay, Software

Journal: bioRxiv

Article Title: Simulated Microgravity Enhances Adipocyte Maturation and Glucose Uptake via Increased Cortical Actin Remodeling

doi: 10.1101/2024.01.30.578049

Figure Lengend Snippet: (a) Ras homolog gene family member A ( RHOA) and Rho-associated coiled-coil kinases ( ROCK1) gene expression was significantly increased in sµg (n=5 biological replicates). (b) Total RhoA protein expression was not affected in 1g and sµg conditions (n=3 biological replicates, the cropped blot is shown, the full-length blot is presented in Supplementary Fig. S1). (c) pCofilin/Cofilin protein expression was increased in sµg conditions (n=6 biological replicates, the cropped blot is shown, the full-length blot is presented in Supplementary Fig. S2). (d) Actin related protein 3 (ARP3) protein expression was significantly increased in sµg (n=3 biological replicates, the cropped blot is shown, the full-length blot is presented in Supplementary Fig. S1). (e) ARP2 protein expression was not affected in 1g and sµg conditions (n=3 biological replicates, the cropped blot is shown, the full-length blot is presented in Supplementary Fig. S2). (f) Simulated microgravity enhances adipocyte maturation via increasing ARP3 protein expression and enhancing cortical actin remodeling. Data are presented as means ± SEM. Comparisons between groups and statistical analysis were performed using an unpaired t-test with two-tailed p-values (*p < 0.05).

Article Snippet: For protein detection, the following primary antibodies (Cell Signaling Technology) were used: rabbit anti-actin related protein 3 (ARP3) (1:1000, Cat# 4738, RRID:AB_2221973), rabbit anti-ARP2 (1.500, Cat# 3128, RRID:AB_2181763), rabbit anti-ras homolog family member A (RhoA) (1:1000, Cat# 2117, RRID:AB_10693922), mouse anti-Akt (1:1000, Cell Signaling, Cat# 2920, RRID:AB_1147620), phosphorylated Akt (pAkt-Ser473) (1:1000 Cat# 4060, RRID:AB_2315049), rabbit anti-cofilin (1:500, Cat# 3318, RRID:AB_2080595), rabbit anti-phosphorylated cofilin (1:500, pcofilin-Ser3, Cat# 3311, RRID:AB_330238), and rabbit anti-beta actin (1:1000, Cat# 4970, RRID:AB_2223172), as a housekeeping protein.

Techniques: Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Simulated Microgravity Enhances Adipocyte Maturation and Glucose Uptake via Increased Cortical Actin Remodeling

doi: 10.1101/2024.01.30.578049

Figure Lengend Snippet: (a) ARP3 expression was significantly increased in mature adipocytes (n=6 biological replicates). (b) pAkt/Akt protein expression was not affected in both conditions after insulin stimulation (n=3 biological replicates, data are normalized to 1g (-ins) condition, cropped blot is shown, the full-length blot is presented in Supplementary Fig. S3). (c) Insulin-stimulated glucose uptake was significantly downregulated in the presence of CK-666 in sµg conditions (n=3 biological replicates). (d) Representative cross-sectional images of adipocytes in the absence (control, top panel) or the presence of CK-666 (bottom panel) in sµg, white arrow points to the brighter cytoplasmic fluorescent signal for GLUT4 in the presence of CK-666 (scale bar-20 µm). (e) GLUT4 translocation to the cell membrane was significantly decreased in the presence of CK-666 (n=4 biological replicates, 10-12 cells were quantified for each replicate). (f) Representative cross-sectional images of adipocytes in the absence (top panel) or the presence of CK-666 (bottom panel) in sµg, white arrow points to the brighter cytoplasmic fluorescent signal for F-actin in the presence of CK-666 (scale bar-20 µm). (g) F-actin cortex to cytoplasm ratio was significantly decreased in the presence of CK-666 (n=3 biological replicates, 10-12 cells were quantified for each replicate). (h) ARP3 inhibition prevented cortical actin remodeling; therefore, GLUT4 translocation to the cell membrane and decreased insulin-stimulated glucose uptake. Data are presented as means ± SEM. Comparisons between groups and statistical analysis were performed using two-way ANOVA with Tukey post hoc test, Mann– Whitney test, or unpaired t-test with two-tailed p-values (*p<0.05, ***p < 0.001, ****p<0.0001).

Article Snippet: For protein detection, the following primary antibodies (Cell Signaling Technology) were used: rabbit anti-actin related protein 3 (ARP3) (1:1000, Cat# 4738, RRID:AB_2221973), rabbit anti-ARP2 (1.500, Cat# 3128, RRID:AB_2181763), rabbit anti-ras homolog family member A (RhoA) (1:1000, Cat# 2117, RRID:AB_10693922), mouse anti-Akt (1:1000, Cell Signaling, Cat# 2920, RRID:AB_1147620), phosphorylated Akt (pAkt-Ser473) (1:1000 Cat# 4060, RRID:AB_2315049), rabbit anti-cofilin (1:500, Cat# 3318, RRID:AB_2080595), rabbit anti-phosphorylated cofilin (1:500, pcofilin-Ser3, Cat# 3311, RRID:AB_330238), and rabbit anti-beta actin (1:1000, Cat# 4970, RRID:AB_2223172), as a housekeeping protein.

Techniques: Expressing, Translocation Assay, Membrane, Inhibition, MANN-WHITNEY, Two Tailed Test

Journal: Seminars in cell & developmental biology

Article Title: Cell polarity and planar cell polarity (PCP) in spermatogenesis

doi: 10.1016/j.semcdb.2017.09.008

Figure Lengend Snippet: Studies have shown that there is a considerable reduction in the expression of Vangl2 on the apical ES in stage VII tubules, which appears to facilitate the conversion of the apical ES at the concave side of spermatid head to a transient ultrastructure known as apical TBC [19, 70, 85] (left panel). Apical TBC represents a giant endocytic vesicle machinery which supports endocytic-mediated protein trafficking event, facilitating protein endocytosis, transcytosis and recycling so that “old” apical ES proteins (e.g., integrins, nectins, laminins, afadin) can be recycled to assemble “new” apical ES when step 8 spermatids appear in stage VIII tubules (right panel). Abbreviations used: Vangl2, Van Gogh-like 2; Arp3, actin-related protein 3; Eps8, epidermal growth factor receptor pathway substrate 8.

Article Snippet: Furthermore, Vangl2 knockdown in the testis perturbed the spatiotemporal expression of actin nucleation protein Arp3 (actin-related protein 3) and actin barbed end capping protein Eps8, facilitating actin filament branching at the apical ES [ 27 ].

Techniques: Expressing

Journal: Endocrinology

Article Title: Coordination of Actin- and Microtubule-Based Cytoskeletons Supports Transport of Spermatids and Residual Bodies/Phagosomes During Spermatogenesis in the Rat Testis

doi: 10.1210/en.2015-1962

Figure Lengend Snippet: Antibodies Used for Various Experiments in This Report

Article Snippet: Antibodies used in this study are listed in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Source and Nature of Antigen Vendor and Catalog Number Host Species and Antibody Nature Working Dilution and Application Arp3 Recombinant human Arp3 protein Sigma-Aldrich, A5979 Mouse monoclonal 1:200 IF EB1 Amino acids 239-268 from the N-terminus, which is located near the C-terminus of EB1 of human origin Santa Cruz Biotechnology, sc-374474 Mouse monoclonal 1:200 IHC EB1 Amino acids 133-202 of peptide fragment from the N-terminus, which is located near the C-terminus of EB1 of human origin Santa Cruz Biotechnology, sc-15347 Rabbit polyclonal 1:300 IF Eps8 Amino acids 628-821 from the N-terminus of mouse Eps8 BD Biosciences, 610143 Mouse monoclonal 1:100 IF p-FAK-Tyr 407 A synthetic phosphopeptide from human FAK containing tyrosine 407 ThermoFisher Scientific, 44-650G Rabbit polyclonal 1:100 IF β1-Integrin Against peptide fragment at N-terminus of β-integrin Santa Cruz Biotechnology, sc-6622 Goat polyclonal 1:100 IF Nectin-3 Against a peptide fragment near the C-terminus of human nectin-3 Santa Cruz Biotechnology, sc-14806 Goat polyclonal 1:100 IF α-Tubulin Full-length native (purified) protein of chicken α-tubulin, but this monoclonal antibody recognized epitope of amino acid residues 426-450 from the N-terminus Abcam, ab7291 Mouse monoclonal 1:500 IHC Open in a separate window Abbreviations: IF, immunofluorescence microscopy; IHC, immunohistochemistry.

Techniques: Recombinant, Purification

Journal: Endocrinology

Article Title: Coordination of Actin- and Microtubule-Based Cytoskeletons Supports Transport of Spermatids and Residual Bodies/Phagosomes During Spermatogenesis in the Rat Testis

doi: 10.1210/en.2015-1962

Figure Lengend Snippet: Adjudin perturbs F-actin organization at the apical ES of spermatids residing at (or near) the luminal edge but also those embedded inside the seminiferous epithelium that fail to undergo exfoliation. Changes in spatiotemporal expression of actin microfilament-organizing proteins, Eps8 and Arp3, effectively disrupted apical ES function at the luminal edge (A) and deep inside the apical compartment (B) at the indicated time points after treatment with adjudin; changes are shown by colocalization of Eps8 (green fluorescence) or Arp3 (green fluorescence) and F-actin (red fluorescence). A, Spatiotemporal expression of Eps8 and Arp3 vs F-actin distribution at stage VII/early VIII apical ES in the normal rat (0 h) was compared with apical ES of spermatids undergoing premature spermiation after 6, 12, and 24 hours of adjudin treatment. Scale bar, 25 μm. B, Spatiotemporal expression of Eps8 or Arp3 vs F-actin at stage VI apical ES in the normal rat (0 h) were compared with apical ES of spermatids embedded inside the epithelium after 6, 12, and 24 hours of adjudin treatment, as indicated by the presence of spermatocytes in these micrographs. Cell nuclei were visualized by 4',6-diamidino-2-phenylindole. Scale bar, 25 μm.

Article Snippet: Antibodies used in this study are listed in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Source and Nature of Antigen Vendor and Catalog Number Host Species and Antibody Nature Working Dilution and Application Arp3 Recombinant human Arp3 protein Sigma-Aldrich, A5979 Mouse monoclonal 1:200 IF EB1 Amino acids 239-268 from the N-terminus, which is located near the C-terminus of EB1 of human origin Santa Cruz Biotechnology, sc-374474 Mouse monoclonal 1:200 IHC EB1 Amino acids 133-202 of peptide fragment from the N-terminus, which is located near the C-terminus of EB1 of human origin Santa Cruz Biotechnology, sc-15347 Rabbit polyclonal 1:300 IF Eps8 Amino acids 628-821 from the N-terminus of mouse Eps8 BD Biosciences, 610143 Mouse monoclonal 1:100 IF p-FAK-Tyr 407 A synthetic phosphopeptide from human FAK containing tyrosine 407 ThermoFisher Scientific, 44-650G Rabbit polyclonal 1:100 IF β1-Integrin Against peptide fragment at N-terminus of β-integrin Santa Cruz Biotechnology, sc-6622 Goat polyclonal 1:100 IF Nectin-3 Against a peptide fragment near the C-terminus of human nectin-3 Santa Cruz Biotechnology, sc-14806 Goat polyclonal 1:100 IF α-Tubulin Full-length native (purified) protein of chicken α-tubulin, but this monoclonal antibody recognized epitope of amino acid residues 426-450 from the N-terminus Abcam, ab7291 Mouse monoclonal 1:500 IHC Open in a separate window Abbreviations: IF, immunofluorescence microscopy; IHC, immunohistochemistry.

Techniques: Expressing, Fluorescence

Journal: Endocrinology

Article Title: Coordination of Actin- and Microtubule-Based Cytoskeletons Supports Transport of Spermatids and Residual Bodies/Phagosomes During Spermatogenesis in the Rat Testis

doi: 10.1210/en.2015-1962

Figure Lengend Snippet: Apical ES function of spermatids located near the luminal edge and those embedded deep inside the epithelium was disrupted after adjudin treatment. Spatiotemporal expression of apical ES integral membrane proteins was examined to confirm changes in F-actin organization that were mediated through disrupted expression of Eps8 and Arp3 at the apical ES. Integral membrane proteins: β1-integrin (a Sertoli cell specific apical ES protein) and nectin-3 (a spermatid specific apical ES protein) vs F-actin were examined at the luminal edge of the apical compartment (A) and within the seminiferous epithelium (B) (as evidenced by the presence of spermatocytes) after treatment with adjudin. At 0 hour, both β1-integrin (red fluorescence) and nectin-3 (red fluorescence) were predominantly expressed on the convex (dorsal) side of the elongated spermatid heads, colocalizing with F-actin (green fluorescence) at the site to confer spermatid adhesion, wherein F-actin was also expressed at the convex side of spermatid heads in this stage VII tubule. Cell nuclei were visualized by 4′,6-diamidino-2-phenylindole. Scale bar, 25 μm (A and B).

Article Snippet: Antibodies used in this study are listed in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibody Source and Nature of Antigen Vendor and Catalog Number Host Species and Antibody Nature Working Dilution and Application Arp3 Recombinant human Arp3 protein Sigma-Aldrich, A5979 Mouse monoclonal 1:200 IF EB1 Amino acids 239-268 from the N-terminus, which is located near the C-terminus of EB1 of human origin Santa Cruz Biotechnology, sc-374474 Mouse monoclonal 1:200 IHC EB1 Amino acids 133-202 of peptide fragment from the N-terminus, which is located near the C-terminus of EB1 of human origin Santa Cruz Biotechnology, sc-15347 Rabbit polyclonal 1:300 IF Eps8 Amino acids 628-821 from the N-terminus of mouse Eps8 BD Biosciences, 610143 Mouse monoclonal 1:100 IF p-FAK-Tyr 407 A synthetic phosphopeptide from human FAK containing tyrosine 407 ThermoFisher Scientific, 44-650G Rabbit polyclonal 1:100 IF β1-Integrin Against peptide fragment at N-terminus of β-integrin Santa Cruz Biotechnology, sc-6622 Goat polyclonal 1:100 IF Nectin-3 Against a peptide fragment near the C-terminus of human nectin-3 Santa Cruz Biotechnology, sc-14806 Goat polyclonal 1:100 IF α-Tubulin Full-length native (purified) protein of chicken α-tubulin, but this monoclonal antibody recognized epitope of amino acid residues 426-450 from the N-terminus Abcam, ab7291 Mouse monoclonal 1:500 IHC Open in a separate window Abbreviations: IF, immunofluorescence microscopy; IHC, immunohistochemistry.

Techniques: Expressing, Fluorescence

Journal: Endocrinology

Article Title: Coordination of Actin- and Microtubule-Based Cytoskeletons Supports Transport of Spermatids and Residual Bodies/Phagosomes During Spermatogenesis in the Rat Testis

doi: 10.1210/en.2015-1962

Figure Lengend Snippet: Antibodies Used for Various Experiments in This Report

Article Snippet: Arp3 , Recombinant human Arp3 protein , Sigma-Aldrich, A5979 , Mouse monoclonal , 1:200 IF.

Techniques: Recombinant, Purification

Journal: Endocrinology

Article Title: Coordination of Actin- and Microtubule-Based Cytoskeletons Supports Transport of Spermatids and Residual Bodies/Phagosomes During Spermatogenesis in the Rat Testis

doi: 10.1210/en.2015-1962

Figure Lengend Snippet: Adjudin perturbs F-actin organization at the apical ES of spermatids residing at (or near) the luminal edge but also those embedded inside the seminiferous epithelium that fail to undergo exfoliation. Changes in spatiotemporal expression of actin microfilament-organizing proteins, Eps8 and Arp3, effectively disrupted apical ES function at the luminal edge (A) and deep inside the apical compartment (B) at the indicated time points after treatment with adjudin; changes are shown by colocalization of Eps8 (green fluorescence) or Arp3 (green fluorescence) and F-actin (red fluorescence). A, Spatiotemporal expression of Eps8 and Arp3 vs F-actin distribution at stage VII/early VIII apical ES in the normal rat (0 h) was compared with apical ES of spermatids undergoing premature spermiation after 6, 12, and 24 hours of adjudin treatment. Scale bar, 25 μm. B, Spatiotemporal expression of Eps8 or Arp3 vs F-actin at stage VI apical ES in the normal rat (0 h) were compared with apical ES of spermatids embedded inside the epithelium after 6, 12, and 24 hours of adjudin treatment, as indicated by the presence of spermatocytes in these micrographs. Cell nuclei were visualized by 4',6-diamidino-2-phenylindole. Scale bar, 25 μm.

Article Snippet: Arp3 , Recombinant human Arp3 protein , Sigma-Aldrich, A5979 , Mouse monoclonal , 1:200 IF.

Techniques: Expressing, Fluorescence

Journal: Endocrinology

Article Title: Coordination of Actin- and Microtubule-Based Cytoskeletons Supports Transport of Spermatids and Residual Bodies/Phagosomes During Spermatogenesis in the Rat Testis

doi: 10.1210/en.2015-1962

Figure Lengend Snippet: Apical ES function of spermatids located near the luminal edge and those embedded deep inside the epithelium was disrupted after adjudin treatment. Spatiotemporal expression of apical ES integral membrane proteins was examined to confirm changes in F-actin organization that were mediated through disrupted expression of Eps8 and Arp3 at the apical ES. Integral membrane proteins: β1-integrin (a Sertoli cell specific apical ES protein) and nectin-3 (a spermatid specific apical ES protein) vs F-actin were examined at the luminal edge of the apical compartment (A) and within the seminiferous epithelium (B) (as evidenced by the presence of spermatocytes) after treatment with adjudin. At 0 hour, both β1-integrin (red fluorescence) and nectin-3 (red fluorescence) were predominantly expressed on the convex (dorsal) side of the elongated spermatid heads, colocalizing with F-actin (green fluorescence) at the site to confer spermatid adhesion, wherein F-actin was also expressed at the convex side of spermatid heads in this stage VII tubule. Cell nuclei were visualized by 4′,6-diamidino-2-phenylindole. Scale bar, 25 μm (A and B).

Article Snippet: Arp3 , Recombinant human Arp3 protein , Sigma-Aldrich, A5979 , Mouse monoclonal , 1:200 IF.

Techniques: Expressing, Fluorescence