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Proteintech dock7 polyclonal antibody

CCN5 promotes LCs senescence by down-regulating RNF213. (A) This table shows the top 10 proteins that combined with CCN5 identified by LC-MS/MS. (B) Bar plot shows the enrichment analysis of proteins that combined with CCN5. (C) The LCs (top panel) and 293t (bottom panel) cells lysates were immunoprecipitated by anti-FLAG antibody (fused with CCN5) or IgG with protein A/G magnetic beads. The precipitated proteins were analyzed by Western blot using <t>anti-DOCK7</t> and anti-RNF213 antibody, respectively. (D) Immunofluorescence co-staining of RNF213 (red) and CCN5 (green) in young and old mice testis. The scale bar represents 100 μm. n = 10 different regions. (E) Scatter plot showing the correlation between CCN5 and RNF213 expression levels in the testicular interstitial region. (F) Western blot shows the level of RNF213 in LCs after recombinant CCN5 protein with CCN5(ΔSP) or CCN5(wt) overexpression. n = 3 technical repeats. (G) Western blot analysis to validate the knockdown efficiency of RNF213 in LCs. n = 3 technical repeats. (H) Western blot shows the fold change of FOXO1, FOXO3, p16, and p21 in LCs after knockdown of RNF213. n = 3 technical repeats. (I) ELISA was used to measure the change of testosterone concentration in the culture supernatant of LCs after knockdown of RNF213. n = 3 technical repeats. (J) SA-β-gal staining of LCs with RNF213 knockdown. The scale bar represents 20 μm. n = 5 technical repeats. (K) Crystal violet staining marks colony formation in LCs with RNF213 knockdown. n = 2 technical repeats. (L) Oil red staining of LCs with RNF213 knockdown. The scale bar represents 10 μm. n = 10 cells.

Dock7 Polyclonal Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/dock7 polyclonal antibody/product/Proteintech
Average 93 stars, based on 7 article reviews

dock7 polyclonal antibody - by Bioz Stars, 2026-04

93/100 stars

Images

1) Product Images from "CCN5 Drives Leydig Cell Aging and Testicular Dysfunction: Insights into Fibrosis, Lipid Dysregulation, and Therapeutic Potential"

Article Title: CCN5 Drives Leydig Cell Aging and Testicular Dysfunction: Insights into Fibrosis, Lipid Dysregulation, and Therapeutic Potential

Journal: Research

doi: 10.34133/research.0762

Figure Legend Snippet: CCN5 promotes LCs senescence by down-regulating RNF213. (A) This table shows the top 10 proteins that combined with CCN5 identified by LC-MS/MS. (B) Bar plot shows the enrichment analysis of proteins that combined with CCN5. (C) The LCs (top panel) and 293t (bottom panel) cells lysates were immunoprecipitated by anti-FLAG antibody (fused with CCN5) or IgG with protein A/G magnetic beads. The precipitated proteins were analyzed by Western blot using anti-DOCK7 and anti-RNF213 antibody, respectively. (D) Immunofluorescence co-staining of RNF213 (red) and CCN5 (green) in young and old mice testis. The scale bar represents 100 μm. n = 10 different regions. (E) Scatter plot showing the correlation between CCN5 and RNF213 expression levels in the testicular interstitial region. (F) Western blot shows the level of RNF213 in LCs after recombinant CCN5 protein with CCN5(ΔSP) or CCN5(wt) overexpression. n = 3 technical repeats. (G) Western blot analysis to validate the knockdown efficiency of RNF213 in LCs. n = 3 technical repeats. (H) Western blot shows the fold change of FOXO1, FOXO3, p16, and p21 in LCs after knockdown of RNF213. n = 3 technical repeats. (I) ELISA was used to measure the change of testosterone concentration in the culture supernatant of LCs after knockdown of RNF213. n = 3 technical repeats. (J) SA-β-gal staining of LCs with RNF213 knockdown. The scale bar represents 20 μm. n = 5 technical repeats. (K) Crystal violet staining marks colony formation in LCs with RNF213 knockdown. n = 2 technical repeats. (L) Oil red staining of LCs with RNF213 knockdown. The scale bar represents 10 μm. n = 10 cells.

Techniques Used: Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Magnetic Beads, Western Blot, Immunofluorescence, Staining, Expressing, Recombinant, Over Expression, Knockdown, Enzyme-linked Immunosorbent Assay, Concentration Assay

Image Search Results

Expression of DOCK7 in the postnatal mouse forebrain. (A) A cartoon representation of the V-SVZ–RMS–OB pathway. (B) Coronal sections of forebrains of P10 mice at positions indicated in the cartoon in A immunostained with antibodies to DOCK7 and DCX and counterstained with DAPI. Boxed regions in III are enlarged and shown at the bottom. Bars: (top) 50 µm; (bottom) 10 µm. (C) Coronal sections of the V-SVZ region of P10 mice immunostained with antibodies to DOCK7 and GFAP, MASH1, or nestin and counterstained with DAPI. Boxed regions are enlarged and shown at the bottom. The yellow dashed line indicates the boundary region between the V-SVZ and the lateral ventricle. Bars: (top) 25 µm; (bottom) 10 µm. (D) Localization of DOCK7 in cultured V-SVZ–derived neuroblasts. Neuroblasts dissociated from V-SVZ tissue of P1–3 mice were fixed at 30 h in vitro, followed by immunostaining with indicated antibodies and counterstaining with DAPI. Bar, 10 µm.

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: Expression of DOCK7 in the postnatal mouse forebrain. (A) A cartoon representation of the V-SVZ–RMS–OB pathway. (B) Coronal sections of forebrains of P10 mice at positions indicated in the cartoon in A immunostained with antibodies to DOCK7 and DCX and counterstained with DAPI. Boxed regions in III are enlarged and shown at the bottom. Bars: (top) 50 µm; (bottom) 10 µm. (C) Coronal sections of the V-SVZ region of P10 mice immunostained with antibodies to DOCK7 and GFAP, MASH1, or nestin and counterstained with DAPI. Boxed regions are enlarged and shown at the bottom. The yellow dashed line indicates the boundary region between the V-SVZ and the lateral ventricle. Bars: (top) 25 µm; (bottom) 10 µm. (D) Localization of DOCK7 in cultured V-SVZ–derived neuroblasts. Neuroblasts dissociated from V-SVZ tissue of P1–3 mice were fixed at 30 h in vitro, followed by immunostaining with indicated antibodies and counterstaining with DAPI. Bar, 10 µm.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-DOCK7 (1:500; ) or rabbit polyclonal Alexa Fluor 488–conjugated anti-DOCK7 (1:200; bs-11825R-A488; Bioss), chicken polyclonal antinestin (1:1,000; NES; Aves Labs), chicken polyclonal anti-GFAP (1:1,000; GFAP; Aves Labs), mouse monoclonal anti-MASH1 (1:2,000; 556604; BD), goat polyclonal anti-DCX (1:1,000; sc-8066; Santa Cruz Biotechnology, Inc.), rat monoclonal anti-BrdU (1:1,000; OBT0030; AbD Serotec), rabbit polyclonal anti-GFP (1:1,000; 598; MBL), and rabbit polyclonal anti-p116 Rip (1:250; NBP1-81035; Novus Biologicals).

Techniques: Expressing, Cell Culture, Derivative Assay, In Vitro, Immunostaining

DOCK7 function is required for the migration of neuroblasts along the RMS. (A) Schematic drawing of postnatal in vivo electroporation. A virtual line (red) connecting the right eye to the craniometrical landmark λ serves as positional marker for plasmid injection into the lateral ventricle (LV). Lateral bars indicate position of electrodes. (B) Composite confocal images of forebrains of mice electroporated at P3 with plasmids expressing EGFP and nontargeting shRNA (scr#1), Dock7-targeting shRNAs (Dock7#1 and Dock7#2), or Dock7#2 shRNA together with DOCK7 cDNA (Dock7#2 + DOCK7) and sacrificed at P10 showing distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway. Slices were counterstained with DAPI. Dotted lines outline borders of sagittal slices. Bar, 500 µm. (C) Quantification of the distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway in the forebrains of mice electroporated at P3 and sacrificed at P10. n = 1,316–2,511 cells from at least three animals for each condition. (D) Quantification of the distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway in the forebrains of mice electroporated at P3 and sacrificed at P17. n = 2,379–2,462 cells from at least three animals for each condition. (E) Enlarged images of neuroblasts in RMSp regions of mice electroporated with indicated constructs at P3 and sacrificed at P10. Arrowheads indicate branching of LP. Bar, 20 µm. (F) Quantification of the length of the LP of neuroblasts in the RMS of mice electroporated with indicated constructs at P3 and sacrificed at P10. n = 91–680 cells from at least three animals for each condition. Data are shown as means ± SEM. **, P < 0.01; ***, P < 0.001; ns, P > 0.05 compared with scr#1; one-way ANOVA with Dunnett’s post hoc test (C and F) or Student’s t test (D).

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: DOCK7 function is required for the migration of neuroblasts along the RMS. (A) Schematic drawing of postnatal in vivo electroporation. A virtual line (red) connecting the right eye to the craniometrical landmark λ serves as positional marker for plasmid injection into the lateral ventricle (LV). Lateral bars indicate position of electrodes. (B) Composite confocal images of forebrains of mice electroporated at P3 with plasmids expressing EGFP and nontargeting shRNA (scr#1), Dock7-targeting shRNAs (Dock7#1 and Dock7#2), or Dock7#2 shRNA together with DOCK7 cDNA (Dock7#2 + DOCK7) and sacrificed at P10 showing distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway. Slices were counterstained with DAPI. Dotted lines outline borders of sagittal slices. Bar, 500 µm. (C) Quantification of the distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway in the forebrains of mice electroporated at P3 and sacrificed at P10. n = 1,316–2,511 cells from at least three animals for each condition. (D) Quantification of the distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway in the forebrains of mice electroporated at P3 and sacrificed at P17. n = 2,379–2,462 cells from at least three animals for each condition. (E) Enlarged images of neuroblasts in RMSp regions of mice electroporated with indicated constructs at P3 and sacrificed at P10. Arrowheads indicate branching of LP. Bar, 20 µm. (F) Quantification of the length of the LP of neuroblasts in the RMS of mice electroporated with indicated constructs at P3 and sacrificed at P10. n = 91–680 cells from at least three animals for each condition. Data are shown as means ± SEM. **, P < 0.01; ***, P < 0.001; ns, P > 0.05 compared with scr#1; one-way ANOVA with Dunnett’s post hoc test (C and F) or Student’s t test (D).

Techniques: Migration, In Vivo, Electroporation, Marker, Plasmid Preparation, Injection, Expressing, shRNA, Transfection, Construct

Knockdown of DOCK7 affects the migration distance, displacement, speed, and LP branching frequency of migrating neuroblasts. (A–F) P2–3 mouse pups were electroporated with plasmids expressing tdTomato and scr#1, Dock7#2 shRNA, or the rescue construct (Dock7#2 + DOCK7). After 5 d, acute sagittal brain slices were prepared, and tdTomato + transfected neuroblasts were imaged by spinning-disk confocal microscopy over a 4-h time period. (A) Time-lapse sequence of scr#1 or Dock7#2 shRNA-expressing cells migrating in the lower vertical arm of the RMSp. Five cells in each slice are labeled in the 0-min panel, and their tracks over time are indicated by lines of the same color. Bars, 70 µm. (B–D) Quantifications of the mean migrated distance (B), displacement (C), and velocity (D) of neuroblasts expressing indicated constructs. n = 50–61 cells from five to seven animals for each condition. (E) Examples of time-lapse series of scr#1 or Dock7#2 shRNA-expressing cells in the lower vertical arm of the RMSp (higher magnification). Arrowheads indicate branching of LP. Bars, 50 µm. (F) Quantification of the number of branching events per h for neuroblasts expressing indicated constructs. n = 50–61 cells from five to seven animals for each condition. Data are shown as means ± SEM. **, P < 0.01; ****, P < 0.0001; ns, P > 0.05 compared with scr#1; one-way ANOVA with Dunnett’s post hoc test.

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: Knockdown of DOCK7 affects the migration distance, displacement, speed, and LP branching frequency of migrating neuroblasts. (A–F) P2–3 mouse pups were electroporated with plasmids expressing tdTomato and scr#1, Dock7#2 shRNA, or the rescue construct (Dock7#2 + DOCK7). After 5 d, acute sagittal brain slices were prepared, and tdTomato + transfected neuroblasts were imaged by spinning-disk confocal microscopy over a 4-h time period. (A) Time-lapse sequence of scr#1 or Dock7#2 shRNA-expressing cells migrating in the lower vertical arm of the RMSp. Five cells in each slice are labeled in the 0-min panel, and their tracks over time are indicated by lines of the same color. Bars, 70 µm. (B–D) Quantifications of the mean migrated distance (B), displacement (C), and velocity (D) of neuroblasts expressing indicated constructs. n = 50–61 cells from five to seven animals for each condition. (E) Examples of time-lapse series of scr#1 or Dock7#2 shRNA-expressing cells in the lower vertical arm of the RMSp (higher magnification). Arrowheads indicate branching of LP. Bars, 50 µm. (F) Quantification of the number of branching events per h for neuroblasts expressing indicated constructs. n = 50–61 cells from five to seven animals for each condition. Data are shown as means ± SEM. **, P < 0.01; ****, P < 0.0001; ns, P > 0.05 compared with scr#1; one-way ANOVA with Dunnett’s post hoc test.

Techniques: Migration, Expressing, shRNA, Construct, Transfection, Confocal Microscopy, Sequencing, Labeling

DOCK7 controls different cellular aspects of neuroblast migration via DHR2/Rac-dependent and R2-dependent pathways. (A) DOCK7 domain structure and deletion and point mutant constructs. ΔR2 comprises the DHR1 domain (amino acids 561–727) and TACC3-binding domain (amino acids 933–1,164). The asterisk indicates the DOCK7(V2022A) mutation. (B) Composite confocal images of forebrains of mice electroporated at P3 with plasmids expressing EGFP and Dock7#2 shRNA + DOCK7, Dock7#2 shRNA + DOCK7ΔDHR2, Dock7#2 shRNA + DOCK7V2022A, or Dock7#2 shRNA + DOCK7ΔR2 and sacrificed at P10, showing distribution of EGFP + transfected neuroblasts along the V-SVZ–RMS–OB pathway. Slices were counterstained with DAPI. Dotted lines outline borders of sagittal slices. Bar, 500 µm. (C) Quantification of the distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway. n = 1,316–1,870 cells from at least three animals for each condition. (D) Quantification of the length of the LP of neuroblasts expressing indicated constructs. n = 91–680 cells from at least three animals for each condition. (E–H) P2–3 mouse pups were electroporated with a tdTomato-expressing plasmid together with one of the indicated constructs. Acute sagittal brain slices were prepared 5 d later and subjected to confocal live-cell imaging. Quantifications of the mean migrated distance (E), displacement (F), velocity (G), and number of branching events per h (H) for the tdTomato + transfected neuroblasts. n = 42–61 cells from four to seven animals for each condition. Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, P > 0.05 compared with scr#1; one-way ANOVA with Dunnett’s post hoc test.

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: DOCK7 controls different cellular aspects of neuroblast migration via DHR2/Rac-dependent and R2-dependent pathways. (A) DOCK7 domain structure and deletion and point mutant constructs. ΔR2 comprises the DHR1 domain (amino acids 561–727) and TACC3-binding domain (amino acids 933–1,164). The asterisk indicates the DOCK7(V2022A) mutation. (B) Composite confocal images of forebrains of mice electroporated at P3 with plasmids expressing EGFP and Dock7#2 shRNA + DOCK7, Dock7#2 shRNA + DOCK7ΔDHR2, Dock7#2 shRNA + DOCK7V2022A, or Dock7#2 shRNA + DOCK7ΔR2 and sacrificed at P10, showing distribution of EGFP + transfected neuroblasts along the V-SVZ–RMS–OB pathway. Slices were counterstained with DAPI. Dotted lines outline borders of sagittal slices. Bar, 500 µm. (C) Quantification of the distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway. n = 1,316–1,870 cells from at least three animals for each condition. (D) Quantification of the length of the LP of neuroblasts expressing indicated constructs. n = 91–680 cells from at least three animals for each condition. (E–H) P2–3 mouse pups were electroporated with a tdTomato-expressing plasmid together with one of the indicated constructs. Acute sagittal brain slices were prepared 5 d later and subjected to confocal live-cell imaging. Quantifications of the mean migrated distance (E), displacement (F), velocity (G), and number of branching events per h (H) for the tdTomato + transfected neuroblasts. n = 42–61 cells from four to seven animals for each condition. Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, P > 0.05 compared with scr#1; one-way ANOVA with Dunnett’s post hoc test.

Techniques: Migration, Mutagenesis, Construct, Binding Assay, Expressing, shRNA, Transfection, Plasmid Preparation, Live Cell Imaging

DOCK7 interacts with p116 Rip . (A) DOCK7 and p116 Rip domain structure. (Top) DOCK7 fragments used in YTH screen/testing. CC, coiled-coil domain; PH, pleckstrin homology domain. (B) YTH interaction between DOCK7-R2 and p116 Rip . Yeast transformed with plasmids expressing DOCK7-R2 or DOCK7-R3 fused to GAL4 DNA–binding domain (GBD) and p116 Rip -C (amino acids 505–933) fused to the GAL4-activation domain (GAD) were grown on medium lacking histidine. Lamin served as negative and H-Ras and phosphatidylinositol 3-OH–kinase-δ (PI 3 KD) as positive controls. (C) Expression of p116 Rip in DCX-positive neuroblasts along the V-SVZ–RMS–OB pathway. Coronal sections of P10 mouse forebrain at positions indicated in the cartoon in immunostained with antibodies to p116 Rip and DCX and counterstained with DAPI. (D) DOCK7–p116 Rip interaction in mammalian cells. Lysates from HEK293 cells transiently expressing Flag-DOCK7 or empty control vector were immunoprecipitated (IP) with an antibody to Flag and analyzed by immunoblotting with antibodies to Flag and p116 Rip . TL, total lysate. (E) GST-p116 Rip -C (amino acids 505–1,024) fusion protein or GST alone (bottom, Coomassie brilliant blue [CBB] staining), immobilized on beads, was incubated with lysates from P5 mouse brains. Bound DOCK7 was detected by immunoblotting with an antibody to DOCK7. (F) Interaction of DOCK7 and p116 Rip in the brain. P10 mouse whole-brain extracts were immunoprecipitated with normal rabbit IgG, an anti-DOCK7 (DOCK7-Ab), or an anti-p116 Rip (p116 Rip -Ab) antibody and then were analyzed by immunoblotting with antibodies to DOCK7 and p116 Rip . Molecular masses are shown in kilodaltons. (G and H) Colocalization of DOCK7 and p116 Rip in V-SVZ–derived neuroblasts. (G) Coronal sections of P10 mouse forebrain at position III in the RMS as indicated in the cartoon in immunostained with antibodies to DOCK7 and p116 Rip and counterstained with DAPI. Boxed regions are enlarged and shown at the bottom. (H) Neuroblasts dissociated from V-SVZ tissue of P1–3 mice were fixed at 30 h in vitro, followed by immunostaining with indicated antibodies and counterstained with DAPI. Bars: (C and G, top) 50 µm; (G, bottom, and H) 10 µm.

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: DOCK7 interacts with p116 Rip . (A) DOCK7 and p116 Rip domain structure. (Top) DOCK7 fragments used in YTH screen/testing. CC, coiled-coil domain; PH, pleckstrin homology domain. (B) YTH interaction between DOCK7-R2 and p116 Rip . Yeast transformed with plasmids expressing DOCK7-R2 or DOCK7-R3 fused to GAL4 DNA–binding domain (GBD) and p116 Rip -C (amino acids 505–933) fused to the GAL4-activation domain (GAD) were grown on medium lacking histidine. Lamin served as negative and H-Ras and phosphatidylinositol 3-OH–kinase-δ (PI 3 KD) as positive controls. (C) Expression of p116 Rip in DCX-positive neuroblasts along the V-SVZ–RMS–OB pathway. Coronal sections of P10 mouse forebrain at positions indicated in the cartoon in immunostained with antibodies to p116 Rip and DCX and counterstained with DAPI. (D) DOCK7–p116 Rip interaction in mammalian cells. Lysates from HEK293 cells transiently expressing Flag-DOCK7 or empty control vector were immunoprecipitated (IP) with an antibody to Flag and analyzed by immunoblotting with antibodies to Flag and p116 Rip . TL, total lysate. (E) GST-p116 Rip -C (amino acids 505–1,024) fusion protein or GST alone (bottom, Coomassie brilliant blue [CBB] staining), immobilized on beads, was incubated with lysates from P5 mouse brains. Bound DOCK7 was detected by immunoblotting with an antibody to DOCK7. (F) Interaction of DOCK7 and p116 Rip in the brain. P10 mouse whole-brain extracts were immunoprecipitated with normal rabbit IgG, an anti-DOCK7 (DOCK7-Ab), or an anti-p116 Rip (p116 Rip -Ab) antibody and then were analyzed by immunoblotting with antibodies to DOCK7 and p116 Rip . Molecular masses are shown in kilodaltons. (G and H) Colocalization of DOCK7 and p116 Rip in V-SVZ–derived neuroblasts. (G) Coronal sections of P10 mouse forebrain at position III in the RMS as indicated in the cartoon in immunostained with antibodies to DOCK7 and p116 Rip and counterstained with DAPI. Boxed regions are enlarged and shown at the bottom. (H) Neuroblasts dissociated from V-SVZ tissue of P1–3 mice were fixed at 30 h in vitro, followed by immunostaining with indicated antibodies and counterstained with DAPI. Bars: (C and G, top) 50 µm; (G, bottom, and H) 10 µm.

Techniques: Transformation Assay, Expressing, Binding Assay, Activation Assay, Plasmid Preparation, Immunoprecipitation, Western Blot, Staining, Incubation, Derivative Assay, In Vitro, Immunostaining

DOCK7 interaction with p116 Rip is essential for neuroblast migration along the RMS. (A) Composite confocal images of forebrains of mice electroporated at P3 with plasmids expressing EGFP and Dock7#2, Dock7#2 + DOCK7, or Dock7#2 + DOCK7Δp116 Rip and sacrificed at P10, showing distribution of EGFP + transfected neuroblasts along the V-SVZ–RMS–OB pathway. Slices were counterstained with DAPI. Dotted lines outline borders of sagittal slices. Bar, 500 µm. (B) Quantification of the distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway. n = 1,316–1,791 cells from at least three animals for each condition. (C) Quantification of the length of the LP of neuroblasts expressing indicated constructs. n = 91–680 cells from at least three animals for each condition. (D–G) P2–3 mouse pups were electroporated with a tdTomato-expressing plasmid together with one of the indicated constructs. Acute sagittal brain slices were prepared 5 d later and subjected to confocal live-cell imaging. Graphs show quantifications of the mean migrated distance (D), displacement (E), velocity (F), and number of branching events per h (G) for the tdTomato + transfected neuroblasts. n = 50–61 cells from five to seven animals for each condition. Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, P > 0.05 compared with scr#1; one-way ANOVA with Dunnett’s post hoc test.

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: DOCK7 interaction with p116 Rip is essential for neuroblast migration along the RMS. (A) Composite confocal images of forebrains of mice electroporated at P3 with plasmids expressing EGFP and Dock7#2, Dock7#2 + DOCK7, or Dock7#2 + DOCK7Δp116 Rip and sacrificed at P10, showing distribution of EGFP + transfected neuroblasts along the V-SVZ–RMS–OB pathway. Slices were counterstained with DAPI. Dotted lines outline borders of sagittal slices. Bar, 500 µm. (B) Quantification of the distribution of EGFP + transfected cells along the V-SVZ–RMS–OB pathway. n = 1,316–1,791 cells from at least three animals for each condition. (C) Quantification of the length of the LP of neuroblasts expressing indicated constructs. n = 91–680 cells from at least three animals for each condition. (D–G) P2–3 mouse pups were electroporated with a tdTomato-expressing plasmid together with one of the indicated constructs. Acute sagittal brain slices were prepared 5 d later and subjected to confocal live-cell imaging. Graphs show quantifications of the mean migrated distance (D), displacement (E), velocity (F), and number of branching events per h (G) for the tdTomato + transfected neuroblasts. n = 50–61 cells from five to seven animals for each condition. Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, P > 0.05 compared with scr#1; one-way ANOVA with Dunnett’s post hoc test.

Techniques: Migration, Expressing, Transfection, Construct, Plasmid Preparation, Live Cell Imaging

DOCK7/p116 Rip signaling controls somal translocation. (A–F) Neuroblasts dissociated from the V-SVZ of P1–3 mice were coelectroporated with one of the indicated constructs and a plasmid coexpressing EGFP and PACT-mKO1, a marker for the centrosome. Cells were reaggregated, embedded in Matrigel, and subjected to confocal live-cell imaging. (A) Examples of time-lapse series showing centrosome and cell soma movements during migration in neuroblasts transfected with indicated constructs. Images are taken from Videos 8–12. Signals of EGFP and PACT-mKO1 are shown in green and red, respectively. White dashed lines indicate advancement of the rear edge of each cell. Arrowheads indicate centrosome position. Bars, 10 µm. (B and C) Temporal changes in the distance between the nucleus and the centrosome (N–C distance; B) and velocity of the cell body (green lines) and the centrosome (red lines; C) in migrating neuroblasts expressing indicated constructs. (D–F) Quantification of velocity (D), somal translocation events (E), and maximum nucleus–centrosome distance (F) for neuroblasts expressing indicated constructs. n = 6 cells (scr#2); n = 5 cells (p116 Rip #1); n = 7 cells (Dock7#2); n = 12 cells (Dock7#2 + DOCK7); and n = 8 cells (Dock7#2 + DOCK7Δp116 Rip ); from 5–12 animals for each condition. Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05; one-way ANOVA with Dunnett’s post hoc test.

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: DOCK7/p116 Rip signaling controls somal translocation. (A–F) Neuroblasts dissociated from the V-SVZ of P1–3 mice were coelectroporated with one of the indicated constructs and a plasmid coexpressing EGFP and PACT-mKO1, a marker for the centrosome. Cells were reaggregated, embedded in Matrigel, and subjected to confocal live-cell imaging. (A) Examples of time-lapse series showing centrosome and cell soma movements during migration in neuroblasts transfected with indicated constructs. Images are taken from Videos 8–12. Signals of EGFP and PACT-mKO1 are shown in green and red, respectively. White dashed lines indicate advancement of the rear edge of each cell. Arrowheads indicate centrosome position. Bars, 10 µm. (B and C) Temporal changes in the distance between the nucleus and the centrosome (N–C distance; B) and velocity of the cell body (green lines) and the centrosome (red lines; C) in migrating neuroblasts expressing indicated constructs. (D–F) Quantification of velocity (D), somal translocation events (E), and maximum nucleus–centrosome distance (F) for neuroblasts expressing indicated constructs. n = 6 cells (scr#2); n = 5 cells (p116 Rip #1); n = 7 cells (Dock7#2); n = 12 cells (Dock7#2 + DOCK7); and n = 8 cells (Dock7#2 + DOCK7Δp116 Rip ); from 5–12 animals for each condition. Data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, P > 0.05; one-way ANOVA with Dunnett’s post hoc test.

Techniques: Translocation Assay, Construct, Plasmid Preparation, Marker, Live Cell Imaging, Migration, Transfection, Expressing

DOCK7/p116 Rip signaling controls somal translocation through F-actin remodeling at the cell rear. (A) Neuroblasts dissociated from the V-SVZ of P1–3 mice were coelectroporated with one of the indicated constructs and a plasmid coexpressing Lifeact-EGFP, a marker for F-actin, and PACT-mKO1. Cells were reaggregated, embedded in Matrigel, and subjected to confocal live-cell imaging. Examples of time-lapse series showing F-actin remodeling during migration in neuroblasts transfected with indicated constructs. Signals of Lifeact-EGFP and PACT-mKO1 are shown in green and red, respectively. White dashed lines indicate advancement of the rear edge (RE) of each cell. White and yellow arrowheads indicate position of centrosome ahead of the nucleus and formation of F-actin condensation at the cell rear, respectively. Linescan profiles to the left of each image show the signal intensities of Lifeact-EGFP (green lines) and PACT-mKO1 (red lines) from the rear edge of the cell soma to the tip of the LP in the corresponding images. Representative images of a total of 5–12 independent experiments/condition are shown. (B) Enrichment of DOCK7 and p116 Rip signals at the cell rear during somal translocation. Reaggregated neuroblasts isolated from V-SVZ tissue of P1–3 mice were embedded in Matrigel and allowed to migrate for 30 h before immunostaining for DOCK7 and p116 Rip . Representative images of neuroblasts captured before (top two rows) and during somal translocation (bottom row) are shown. Enrichment of DOCK7 and p116 Rip signals at the cell rear just before and during somal translocation is indicated by arrowheads. Bars, 10 µm.

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Figure Lengend Snippet: DOCK7/p116 Rip signaling controls somal translocation through F-actin remodeling at the cell rear. (A) Neuroblasts dissociated from the V-SVZ of P1–3 mice were coelectroporated with one of the indicated constructs and a plasmid coexpressing Lifeact-EGFP, a marker for F-actin, and PACT-mKO1. Cells were reaggregated, embedded in Matrigel, and subjected to confocal live-cell imaging. Examples of time-lapse series showing F-actin remodeling during migration in neuroblasts transfected with indicated constructs. Signals of Lifeact-EGFP and PACT-mKO1 are shown in green and red, respectively. White dashed lines indicate advancement of the rear edge (RE) of each cell. White and yellow arrowheads indicate position of centrosome ahead of the nucleus and formation of F-actin condensation at the cell rear, respectively. Linescan profiles to the left of each image show the signal intensities of Lifeact-EGFP (green lines) and PACT-mKO1 (red lines) from the rear edge of the cell soma to the tip of the LP in the corresponding images. Representative images of a total of 5–12 independent experiments/condition are shown. (B) Enrichment of DOCK7 and p116 Rip signals at the cell rear during somal translocation. Reaggregated neuroblasts isolated from V-SVZ tissue of P1–3 mice were embedded in Matrigel and allowed to migrate for 30 h before immunostaining for DOCK7 and p116 Rip . Representative images of neuroblasts captured before (top two rows) and during somal translocation (bottom row) are shown. Enrichment of DOCK7 and p116 Rip signals at the cell rear just before and during somal translocation is indicated by arrowheads. Bars, 10 µm.

Techniques: Translocation Assay, Construct, Plasmid Preparation, Marker, Live Cell Imaging, Migration, Transfection, Isolation, Immunostaining

Journal: Research

Article Title: CCN5 Drives Leydig Cell Aging and Testicular Dysfunction: Insights into Fibrosis, Lipid Dysregulation, and Therapeutic Potential

doi: 10.34133/research.0762

Figure Lengend Snippet: CCN5 promotes LCs senescence by down-regulating RNF213. (A) This table shows the top 10 proteins that combined with CCN5 identified by LC-MS/MS. (B) Bar plot shows the enrichment analysis of proteins that combined with CCN5. (C) The LCs (top panel) and 293t (bottom panel) cells lysates were immunoprecipitated by anti-FLAG antibody (fused with CCN5) or IgG with protein A/G magnetic beads. The precipitated proteins were analyzed by Western blot using anti-DOCK7 and anti-RNF213 antibody, respectively. (D) Immunofluorescence co-staining of RNF213 (red) and CCN5 (green) in young and old mice testis. The scale bar represents 100 μm. n = 10 different regions. (E) Scatter plot showing the correlation between CCN5 and RNF213 expression levels in the testicular interstitial region. (F) Western blot shows the level of RNF213 in LCs after recombinant CCN5 protein with CCN5(ΔSP) or CCN5(wt) overexpression. n = 3 technical repeats. (G) Western blot analysis to validate the knockdown efficiency of RNF213 in LCs. n = 3 technical repeats. (H) Western blot shows the fold change of FOXO1, FOXO3, p16, and p21 in LCs after knockdown of RNF213. n = 3 technical repeats. (I) ELISA was used to measure the change of testosterone concentration in the culture supernatant of LCs after knockdown of RNF213. n = 3 technical repeats. (J) SA-β-gal staining of LCs with RNF213 knockdown. The scale bar represents 20 μm. n = 5 technical repeats. (K) Crystal violet staining marks colony formation in LCs with RNF213 knockdown. n = 2 technical repeats. (L) Oil red staining of LCs with RNF213 knockdown. The scale bar represents 10 μm. n = 10 cells.

Article Snippet: The following primary antibodies were used for immunoblotting: DYKDDDDK tag Monoclonal antibody (Proteintech, 66008-4-IG, 1:5,000), RNF213 Antibody (Novus, NBP1-88466, 1:1,000), Smad2 (D43B4) XP Rabbit mAb (CST, 5339T, 1:1,000), Phospho-SMAD2 (Ser465/Ser467) (E8F3R) Rabbit mAb (CST, 4511T, 1:1,000), Smad3 Rabbit pAb (Abclonal, A11471, 1:1,000), Phospho-Smad3-S423/S425 Rabbit mAb (Abclonal, AP0727, 1:1,000), alpha-SMA Antibody (Affinity Biosciences, AF1032, 1:1,000), ccn5-rabbit-polyclonal-antibody (Origene, TA383325; 1:1,000), and DOCK7 Polyclonal antibody (proteintech, 13000-1-AP, 1:1,000).

Techniques: Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Magnetic Beads, Western Blot, Immunofluorescence, Staining, Expressing, Recombinant, Over Expression, Knockdown, Enzyme-linked Immunosorbent Assay, Concentration Assay

DOCK7 is required for replication stress response. ( A, C, E ) Survival assays of control and DOCK7-depleted U2OS cells treated with indicated doses of IR, HU or CPT. Data are represented as the mean ± SEM of n = 3 independent experiments. ( B, D, F ) Phosphorylation of CHK1 and CHK2 were determined by immunoblotting in control and DOCK7-depleted U2OS cells treated with 10 Gy IR, 10 mM HU or 1 μM CPT for 2 h. ( G-J ) U2OS cells were labeled with 50 μM IdU, and then treated with or without HU, thereafter incubated with 200 μM CldU for indicated time, the fork speed and the length of CIdU track in control and DOCK7-depleted cells were determined by measuring the length of CIdU track panels (H and J), representative pictures of fibers are shown in panels (G and I). The graphs represent mean ± S.D., two-tailed, unpaired t -test. ***p<0.001. ( K ) ER-AsiSI U2OS cells were transfected with vector control or FLAG-DOCK7 for 36 h before being treated with or without 1 μM 4-OHT for 4 h. After cells were harvested, ChIP experiments were performed using FLAG antibody. Error bars represent SEM from three independent experiments. ***p<0.001.

Journal: Nucleic Acids Research

Article Title: DOCK7 protects against replication stress by promoting RPA stability on chromatin

doi: 10.1093/nar/gkab134

Figure Lengend Snippet: DOCK7 is required for replication stress response. ( A, C, E ) Survival assays of control and DOCK7-depleted U2OS cells treated with indicated doses of IR, HU or CPT. Data are represented as the mean ± SEM of n = 3 independent experiments. ( B, D, F ) Phosphorylation of CHK1 and CHK2 were determined by immunoblotting in control and DOCK7-depleted U2OS cells treated with 10 Gy IR, 10 mM HU or 1 μM CPT for 2 h. ( G-J ) U2OS cells were labeled with 50 μM IdU, and then treated with or without HU, thereafter incubated with 200 μM CldU for indicated time, the fork speed and the length of CIdU track in control and DOCK7-depleted cells were determined by measuring the length of CIdU track panels (H and J), representative pictures of fibers are shown in panels (G and I). The graphs represent mean ± S.D., two-tailed, unpaired t -test. ***p<0.001. ( K ) ER-AsiSI U2OS cells were transfected with vector control or FLAG-DOCK7 for 36 h before being treated with or without 1 μM 4-OHT for 4 h. After cells were harvested, ChIP experiments were performed using FLAG antibody. Error bars represent SEM from three independent experiments. ***p<0.001.

Article Snippet: Anti-DOCK6 (amino acids 2026–2047; 1:1000) and anti-DOCK7 (amino acids 2110–2132; 1:1000) rabbit polyclonal antibodies were generated at Cocalico Biologicals Inc. (Reamstown, PA) using the indicated KLH-conjugates peptides; Anti-RPA1 was purchased (A300–241A, 1:5000) from Bethyl Laboratories.

Techniques: Western Blot, Labeling, Incubation, Two Tailed Test, Transfection, Plasmid Preparation

$DOCK7 is phosphorylated by ATR and then recruited to chromatin by MDC1 to regulate replication stress response. ( A and H ) HEK293T cells stably transfected with HA-MDC1 (A), FLAG-DOCK7 WT or S1438A mutant (H) were incubated with 10 μM EdU for 20 min before treated or untreated with HU. Replication fork recruited proteins were isolated by iPOND and blotted with indicated antibodies. ( B and E ) Control and MDC1-depleted or ATR-depleted U2OS cells were treated with 10 mM HU for 1 h, cells were then harvested and separated into chromatin and soluble fractions, the protein level of DOCK7 in each fraction was detected by immunoblotting assay. ( C ) HEK293T cells were transfected with indicated MDC1 constructs for 24 h, co-immunoprecipitation (co-IP) assay were performed using anti-HA agarose beads and then blotted with indicated antibodies. ( D ) HEK293T cells transfected with FLAG-DOCK7 were pre-treated with DMSO or 50 nM VX-970 for 2 h then treated 10 mM HU for 2 h, cell lysates were immunoprecipitated with anti-FLAG agarose beads, and left untreated or were treated with phosphatase, cell lysates were blotted with the indicated antibodies. ( F ) HEK293T cells transfected with WT or S1438A mutant of DOCK7 were incubated with 10 mM HU for 1 h, cell lysates were then immunoprecipitated with anti-FLAG agarose beads and blotted with indicated antibodies. ( G ) The protein levels of FLAG-DOCK7 in chromatin and soluble fractions of HEK293T cells transfected with WT or S1438A mutant of FLAG-DOCK7 before or after HU treatment were detected by immunoblotting assay. ( I ) Lysates from HEK293T cells transfected with WT or S1438A mutant of DOCK7 with or without HU treatment were used in a PAK-CRIB pull-down assay. The immunoprecipitates were subjected to immunoblotting with the indicated antibodies. ( J ) The survival rate of control or DOCK7-depleted U2OS cells transfected vector control or indicated DOCK7 constructs were assessed by colony formation assay. Error bars represent SEM from three independent experiments.$

Journal: Nucleic Acids Research

Article Title: DOCK7 protects against replication stress by promoting RPA stability on chromatin

doi: 10.1093/nar/gkab134

Figure Lengend Snippet: DOCK7 is phosphorylated by ATR and then recruited to chromatin by MDC1 to regulate replication stress response. ( A and H ) HEK293T cells stably transfected with HA-MDC1 (A), FLAG-DOCK7 WT or S1438A mutant (H) were incubated with 10 μM EdU for 20 min before treated or untreated with HU. Replication fork recruited proteins were isolated by iPOND and blotted with indicated antibodies. ( B and E ) Control and MDC1-depleted or ATR-depleted U2OS cells were treated with 10 mM HU for 1 h, cells were then harvested and separated into chromatin and soluble fractions, the protein level of DOCK7 in each fraction was detected by immunoblotting assay. ( C ) HEK293T cells were transfected with indicated MDC1 constructs for 24 h, co-immunoprecipitation (co-IP) assay were performed using anti-HA agarose beads and then blotted with indicated antibodies. ( D ) HEK293T cells transfected with FLAG-DOCK7 were pre-treated with DMSO or 50 nM VX-970 for 2 h then treated 10 mM HU for 2 h, cell lysates were immunoprecipitated with anti-FLAG agarose beads, and left untreated or were treated with phosphatase, cell lysates were blotted with the indicated antibodies. ( F ) HEK293T cells transfected with WT or S1438A mutant of DOCK7 were incubated with 10 mM HU for 1 h, cell lysates were then immunoprecipitated with anti-FLAG agarose beads and blotted with indicated antibodies. ( G ) The protein levels of FLAG-DOCK7 in chromatin and soluble fractions of HEK293T cells transfected with WT or S1438A mutant of FLAG-DOCK7 before or after HU treatment were detected by immunoblotting assay. ( I ) Lysates from HEK293T cells transfected with WT or S1438A mutant of DOCK7 with or without HU treatment were used in a PAK-CRIB pull-down assay. The immunoprecipitates were subjected to immunoblotting with the indicated antibodies. ( J ) The survival rate of control or DOCK7-depleted U2OS cells transfected vector control or indicated DOCK7 constructs were assessed by colony formation assay. Error bars represent SEM from three independent experiments.

Techniques: Stable Transfection, Transfection, Mutagenesis, Incubation, Isolation, Western Blot, Construct, Co-Immunoprecipitation Assay, Immunoprecipitation, Pull Down Assay, Plasmid Preparation, Colony Assay

$DOCK7 increases the protein stability of RPA1 in chromatin and replication fork. ( A ) The distribution of indicated proteins in the chromatin and soluble fractions of control or DOCK7-depleted U2OS cells after treated with 10 mM HU for 2 h were determined by immunoblotting assay. ( B and C ) Representative images (B) and quantification (C) of RPA2 foci. More than 200 cells were counted in each experiment. Error bars represent SEM from three independent experiments. ***p<0.001. ( D ) Control and DOCK7-depleted HEK293T cells were incubated with 10 μM EdU for 20 min before or after HU treatment. Replication fork recruited proteins were isolated by iPOND and blotted with indicated antibodies. ( E ) Chromatin and soluble fraction of cell lysates separated from MG132-treated HEK293T were blotted to measure the expression level of RPA1. ( F ) The protein contents of RPA1 in the chromatin and soluble fraction of control or DOCK7-depleted HEK293T cells treated with 20 μM CHX for different time points were detected by immunoblotting assay. ( G ) Control or DOCK7-depleted HEK293T cells were transfected with FLAG-RPA1 and His-Ub for 24 h before being treated with 10 mM HU for 1 h, the chromatin and soluble fractions of harvested cell lysates were then immunoprecipitated with nickel (His) beads and blots were probed with indicated antibodies.$

Journal: Nucleic Acids Research

Article Title: DOCK7 protects against replication stress by promoting RPA stability on chromatin

doi: 10.1093/nar/gkab134

Figure Lengend Snippet: DOCK7 increases the protein stability of RPA1 in chromatin and replication fork. ( A ) The distribution of indicated proteins in the chromatin and soluble fractions of control or DOCK7-depleted U2OS cells after treated with 10 mM HU for 2 h were determined by immunoblotting assay. ( B and C ) Representative images (B) and quantification (C) of RPA2 foci. More than 200 cells were counted in each experiment. Error bars represent SEM from three independent experiments. ***p<0.001. ( D ) Control and DOCK7-depleted HEK293T cells were incubated with 10 μM EdU for 20 min before or after HU treatment. Replication fork recruited proteins were isolated by iPOND and blotted with indicated antibodies. ( E ) Chromatin and soluble fraction of cell lysates separated from MG132-treated HEK293T were blotted to measure the expression level of RPA1. ( F ) The protein contents of RPA1 in the chromatin and soluble fraction of control or DOCK7-depleted HEK293T cells treated with 20 μM CHX for different time points were detected by immunoblotting assay. ( G ) Control or DOCK7-depleted HEK293T cells were transfected with FLAG-RPA1 and His-Ub for 24 h before being treated with 10 mM HU for 1 h, the chromatin and soluble fractions of harvested cell lysates were then immunoprecipitated with nickel (His) beads and blots were probed with indicated antibodies.

Techniques: Western Blot, Incubation, Isolation, Expressing, Transfection, Immunoprecipitation

$DOCK7-Rac1/Cdc42-PAK1 pathway is critical for replication stress response through regulating the chromatin recruitment of RPA1. ( A-C ) Control or DOCK7-depleted U2OS cells were transfected with vector control, WT DOCK7 or DOCK7ΔDHR2 truncates for 24 h before treated with 10 mM HU for 1 h. The expression levels of RPA1 and RPA2 in the chromatin and soluble fraction of harvested cells were determined by immunoblotting assay (A); RPA2 foci formation was detected by immunofluorescence (B) and quantified (C). More than 200 cells were counted in each experiment. Error bars represent SEM from three independent experiments. ***p<0.001. ( D ) Survival assays of control or DOCK7-depleted U2OS cells transfected with vector control, WT DOCK7 or DOCK7ΔDHR2 in response to HU. Error bars represent SEM from three independent experiments. ( E ) HEK293T cells were transfected with vector control, WT DOCK7 or DOCK7ΔDHR2 for 24 h before treatment with 10 mM HU for 2 h, cells lysates were then used in a GST-PAK-CRIB pull-down assay to detect the activation of Rac1 and Cdc42. ( F and I ) Chromatin and soluble fractions of cell lysates derived from DMSO or 1 μM R-ketorolac pretreatment (F) and control or PAK1 knockdown (I) U2OS cells were subjected to immunoblot with the indicated antibodies. ( G, H , J and K ) Representative images (G and J) and quantification (H and K) of RPA2 foci in DMSO or R-ketorolac pretreatment (G and H) and control or PAK1 knockdown (J and K) U2OS cells after HU treatment were detected and analyzed by immunofluorescence. ( L ) RPA1 and RPA2 proteins isolated by iPOND from control or PAK1 depletion HEK293T cells were detected by immunoblotting assay. ( M and N ) DNA fiber assay was performed to detect the length of CIdU track after HU was removed in control or PAK1-depleted U2OS cells (N), representative pictures of fibers are shown in (M). The graphs represent mean ± S.D., two-tailed, unpaired t -test. ***p<0.001.$

Journal: Nucleic Acids Research

Article Title: DOCK7 protects against replication stress by promoting RPA stability on chromatin

doi: 10.1093/nar/gkab134

Figure Lengend Snippet: DOCK7-Rac1/Cdc42-PAK1 pathway is critical for replication stress response through regulating the chromatin recruitment of RPA1. ( A-C ) Control or DOCK7-depleted U2OS cells were transfected with vector control, WT DOCK7 or DOCK7ΔDHR2 truncates for 24 h before treated with 10 mM HU for 1 h. The expression levels of RPA1 and RPA2 in the chromatin and soluble fraction of harvested cells were determined by immunoblotting assay (A); RPA2 foci formation was detected by immunofluorescence (B) and quantified (C). More than 200 cells were counted in each experiment. Error bars represent SEM from three independent experiments. ***p<0.001. ( D ) Survival assays of control or DOCK7-depleted U2OS cells transfected with vector control, WT DOCK7 or DOCK7ΔDHR2 in response to HU. Error bars represent SEM from three independent experiments. ( E ) HEK293T cells were transfected with vector control, WT DOCK7 or DOCK7ΔDHR2 for 24 h before treatment with 10 mM HU for 2 h, cells lysates were then used in a GST-PAK-CRIB pull-down assay to detect the activation of Rac1 and Cdc42. ( F and I ) Chromatin and soluble fractions of cell lysates derived from DMSO or 1 μM R-ketorolac pretreatment (F) and control or PAK1 knockdown (I) U2OS cells were subjected to immunoblot with the indicated antibodies. ( G, H , J and K ) Representative images (G and J) and quantification (H and K) of RPA2 foci in DMSO or R-ketorolac pretreatment (G and H) and control or PAK1 knockdown (J and K) U2OS cells after HU treatment were detected and analyzed by immunofluorescence. ( L ) RPA1 and RPA2 proteins isolated by iPOND from control or PAK1 depletion HEK293T cells were detected by immunoblotting assay. ( M and N ) DNA fiber assay was performed to detect the length of CIdU track after HU was removed in control or PAK1-depleted U2OS cells (N), representative pictures of fibers are shown in (M). The graphs represent mean ± S.D., two-tailed, unpaired t -test. ***p<0.001.

Techniques: Transfection, Plasmid Preparation, Expressing, Western Blot, Immunofluorescence, Pull Down Assay, Activation Assay, Derivative Assay, Isolation, Two Tailed Test

$RPA1 phosphorylated by PAK1 at Ser-135 and Thr-180 is critical for its role in replication stress response. ( A–C ) Control or DOCK7-depleted HEK293T cells were transfected with indicated FLAG-RPA1 constructs for 24 h. Cells were then pretreated with or without inhibitors (R-ketorolac or PAK1 inhibitor) before or after HU treatment. FLAG-RPA1 was coimmunoprecipitated from cell lysates and loaded on both normal and Phospho-tag gel, thereafter blotted with indicated antibodies. ( D ) Purified WT and ST/A mutant of RPA1 were incubated with or without constitutively active PAK1 (50 aa-150 aa) and incubated with γ- [32P] ATP for 30 min at 30°C before subjected to autoradiography. ( E–G ) Control or RPA1-depleted U2OS cells were transfected with WT or ST/A mutant of FLAG-RPA1 for 24 h before treatment with 10 mM HU for 1 h, and then the distribution of RPA1 and RPA2 in the chromatin and soluble fractions of cells were determined by immunoblotting (E). Representative images (F) and quantification (G) of RPA2 foci were analyzed by immunofluorescence. More than 200 cells were counted in each experiment. Error bars represent SEM from three independent experiments. ***p<0.001. ( H–J ) RPA1-depleted cells were transfected with WT or ST/A mutant of FLAG-RPA1 for 24 h before HU treatment, and then IPOND assay in HEK293T cells was performed to detect the distribution of the WT or ST/A mutant of RPA1 on the replication fork (H). DNA fiber assay in U2OS cells was performed to detect the length of CIdU track after HU was removed (J), and the representative pictures are shown in panel (I). The graphs represent mean ± S.D., two-tailed, unpaired t -test. ***p<0.001. ( K ) Control or RPA1-depleted U2OS cells were transfected with vector control, WT or ST/A mutant of FLAG-RPA1 for 24 h, treated with the indicated concentrations of HU and survival was measured using the colony formation assay. Error bars represent SEM from three independent experiments. ( L ) The protein stability of WT or ST/A mutant of RPA1 in the chromatin and soluble fraction of RPA1-depleted HEK293T cells under CHX treatment were analyzed by immunoblotting assay. ( M ) RPA1-depleted HEK293T cells were transfected with WT or ST/A mutant of FLAG-RPA1 and His-Ub for 24 h before HU treatment. Chromatin and soluble fractions derived from harvested cells were immunoprecipitated with nickel (His) beads and then blotted with indicated antibodies$

Journal: Nucleic Acids Research

Article Title: DOCK7 protects against replication stress by promoting RPA stability on chromatin

doi: 10.1093/nar/gkab134

Figure Lengend Snippet: RPA1 phosphorylated by PAK1 at Ser-135 and Thr-180 is critical for its role in replication stress response. ( A–C ) Control or DOCK7-depleted HEK293T cells were transfected with indicated FLAG-RPA1 constructs for 24 h. Cells were then pretreated with or without inhibitors (R-ketorolac or PAK1 inhibitor) before or after HU treatment. FLAG-RPA1 was coimmunoprecipitated from cell lysates and loaded on both normal and Phospho-tag gel, thereafter blotted with indicated antibodies. ( D ) Purified WT and ST/A mutant of RPA1 were incubated with or without constitutively active PAK1 (50 aa-150 aa) and incubated with γ- [32P] ATP for 30 min at 30°C before subjected to autoradiography. ( E–G ) Control or RPA1-depleted U2OS cells were transfected with WT or ST/A mutant of FLAG-RPA1 for 24 h before treatment with 10 mM HU for 1 h, and then the distribution of RPA1 and RPA2 in the chromatin and soluble fractions of cells were determined by immunoblotting (E). Representative images (F) and quantification (G) of RPA2 foci were analyzed by immunofluorescence. More than 200 cells were counted in each experiment. Error bars represent SEM from three independent experiments. ***p<0.001. ( H–J ) RPA1-depleted cells were transfected with WT or ST/A mutant of FLAG-RPA1 for 24 h before HU treatment, and then IPOND assay in HEK293T cells was performed to detect the distribution of the WT or ST/A mutant of RPA1 on the replication fork (H). DNA fiber assay in U2OS cells was performed to detect the length of CIdU track after HU was removed (J), and the representative pictures are shown in panel (I). The graphs represent mean ± S.D., two-tailed, unpaired t -test. ***p<0.001. ( K ) Control or RPA1-depleted U2OS cells were transfected with vector control, WT or ST/A mutant of FLAG-RPA1 for 24 h, treated with the indicated concentrations of HU and survival was measured using the colony formation assay. Error bars represent SEM from three independent experiments. ( L ) The protein stability of WT or ST/A mutant of RPA1 in the chromatin and soluble fraction of RPA1-depleted HEK293T cells under CHX treatment were analyzed by immunoblotting assay. ( M ) RPA1-depleted HEK293T cells were transfected with WT or ST/A mutant of FLAG-RPA1 and His-Ub for 24 h before HU treatment. Chromatin and soluble fractions derived from harvested cells were immunoprecipitated with nickel (His) beads and then blotted with indicated antibodies

Techniques: Transfection, Construct, Purification, Mutagenesis, Incubation, Autoradiography, Western Blot, Immunofluorescence, Two Tailed Test, Plasmid Preparation, Colony Assay, Derivative Assay, Immunoprecipitation

DOCK7 depletion enhances chemotherapy of ovarian cancer cells in vitro and in vivo . ( A ) Cell survival rate after CPT treatment was determined by colony formation assay in control or DOCK7-depleted OVCAR8 cells. Error bars represent SEM from three independent experiments. ( B–D ) Control or DOCK7-depleted OVCAR8 cells were subcutaneously injected into the flank of NOD-SCID mice. Mice were treated with saline or CPT (10 mg/kg i.p. 3 days × 8 times). Tumor images were shown in (B), and tumor weight (C) and volume (D) were assessed. Data points (mean ± SEM) are shown from five biologically independent samples by two-sided unpaired t test. ( E ) Working model of RPA1 phosphorylation regulated by DOCK7 signaling. DOCK7 is phosphorylated by ATR during replication stress and then recruited to the chromatin and DNA damage sites by MDC1. Thereafter, DOCK7 facilitates the GTP-loading of Rac1/Cdc42, which in turn activate PAK1 to phosphorylate and stabilize chromatin-loaded RPA1 to stabilize and enable replication fork restart.

Journal: Nucleic Acids Research

Article Title: DOCK7 protects against replication stress by promoting RPA stability on chromatin

doi: 10.1093/nar/gkab134

Figure Lengend Snippet: DOCK7 depletion enhances chemotherapy of ovarian cancer cells in vitro and in vivo . ( A ) Cell survival rate after CPT treatment was determined by colony formation assay in control or DOCK7-depleted OVCAR8 cells. Error bars represent SEM from three independent experiments. ( B–D ) Control or DOCK7-depleted OVCAR8 cells were subcutaneously injected into the flank of NOD-SCID mice. Mice were treated with saline or CPT (10 mg/kg i.p. 3 days × 8 times). Tumor images were shown in (B), and tumor weight (C) and volume (D) were assessed. Data points (mean ± SEM) are shown from five biologically independent samples by two-sided unpaired t test. ( E ) Working model of RPA1 phosphorylation regulated by DOCK7 signaling. DOCK7 is phosphorylated by ATR during replication stress and then recruited to the chromatin and DNA damage sites by MDC1. Thereafter, DOCK7 facilitates the GTP-loading of Rac1/Cdc42, which in turn activate PAK1 to phosphorylate and stabilize chromatin-loaded RPA1 to stabilize and enable replication fork restart.

Techniques: In Vitro, In Vivo, Colony Assay, Injection

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Expressing, Cell Culture, Derivative Assay, In Vitro, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Migration, In Vivo, Electroporation, Marker, Plasmid Preparation, Injection, Expressing, shRNA, Transfection, Construct

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Migration, Expressing, shRNA, Construct, Transfection, Confocal Microscopy, Sequencing, Labeling

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Migration, Mutagenesis, Construct, Binding Assay, Expressing, shRNA, Transfection, Plasmid Preparation, Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Migration, Expressing, Transfection, Construct, Plasmid Preparation, Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Translocation Assay, Construct, Plasmid Preparation, Marker, Live Cell Imaging, Migration, Transfection, Expressing

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Translocation Assay, Construct, Plasmid Preparation, Marker, Live Cell Imaging, Migration, Transfection, Isolation, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-GFP (1:1,000; 598; MBL), mouse monoclonal anti–βIII-tubulin/Tuj1 (1:2,000; MMS-435P; Covance), rabbit polyclonal anti-DOCK7 Alexa Fluor 488–conjugated (1:200; bs-11825R-A488; Bioss), goat polyclonal anti-DCX (1:1,000; sc-8066; Santa Cruz Biotechnology, Inc.), and rabbit polyclonal anti-p116 Rip (1:250; NBP1-81035; Novus Biologicals).

Techniques: Expressing, Cell Culture, Derivative Assay, In Vitro, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Migration, In Vivo, Electroporation, Marker, Plasmid Preparation, Injection, Expressing, shRNA, Transfection, Construct

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Migration, Expressing, shRNA, Construct, Transfection, Confocal Microscopy, Sequencing, Labeling

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Migration, Mutagenesis, Construct, Binding Assay, Expressing, shRNA, Transfection, Plasmid Preparation, Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Migration, Expressing, Transfection, Construct, Plasmid Preparation, Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Translocation Assay, Construct, Plasmid Preparation, Marker, Live Cell Imaging, Migration, Transfection, Expressing

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Translocation Assay, Construct, Plasmid Preparation, Marker, Live Cell Imaging, Migration, Transfection, Isolation, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Article Snippet: For Western blots depicted in Fig. S1 (A and B), tissues prepared from the V-SVZ and RMSp as well as the OB and RMSa of P10 and P45 CD1 mice were snap-frozen in liquid nitrogen, sonicated in 1% SDS buffer, and subjected to Western blot analysis with rabbit polyclonal anti-DOCK7 antibody (1:1,000; ) and rabbit monoclonal anti-GAPDH antibody (1:1,000; 5174; Cell Signaling Technology) as a loading control.

Techniques: Expressing, Cell Culture, Derivative Assay, In Vitro, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Migration, In Vivo, Electroporation, Marker, Plasmid Preparation, Injection, Expressing, shRNA, Transfection, Construct

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Knockdown, Migration, Expressing, shRNA, Construct, Transfection, Confocal Microscopy, Sequencing, Labeling

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Migration, Mutagenesis, Construct, Binding Assay, Expressing, shRNA, Transfection, Plasmid Preparation, Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Transformation Assay, Expressing, Binding Assay, Activation Assay, Control, Plasmid Preparation, Immunoprecipitation, Western Blot, Staining, Incubation, Derivative Assay, In Vitro, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Migration, Expressing, Transfection, Construct, Plasmid Preparation, Live Cell Imaging

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Translocation Assay, Construct, Plasmid Preparation, Marker, Live Cell Imaging, Migration, Transfection, Expressing

Journal: The Journal of Cell Biology

Article Title: Dual role for DOCK7 in tangential migration of interneuron precursors in the postnatal forebrain

doi: 10.1083/jcb.201704157

Techniques: Translocation Assay, Construct, Plasmid Preparation, Marker, Live Cell Imaging, Migration, Transfection, Isolation, Immunostaining

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