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

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).

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: 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

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.

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: 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

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.

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: 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

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.

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: Transformation Assay, Expressing, Binding Assay, Activation Assay, 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

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.

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: 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

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.

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: 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

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.

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

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) The Cancer Genome Atlas (TGCA) expression profile for Dock-C family members, Dock6, Dock7, and Dock8, in triple-negative breast cancer and normal tissue. ( B ) Protein expression of Dock7 in a breast cancer cell-line cohort. ( C ) Dock7 mRNA levels across patients with either receptor-positive (BC) or triple-negative breast cancers (TNBC). ( D and E ) Quantification of colonies formed in soft agar suspension for SKRB3 and MCF7, respectively. (Below) Western blot showing Dock7 protein expression after either control or Dock7 KD for each cell line. ( F ) Dock7 expression profile for MDA-MB-231 cells that were used for either (G) soft agar assay or ( H ) survival in serum-free media for 4 days. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in ( C ), ( D - E ), and ( G - H ) represent means ± SD; ***p < 0.001, and **p < 0.01

Article Snippet: Dock7 antibody was purchased from Santa Cruz Biotechnology, PHLPP was purchased from Proteintech, and all other antibodies were obtained from Cell Signaling Technologies.

Techniques: Expressing, Suspension, Western Blot, Control, Soft Agar Assay

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A and B ) Soft agar colony formation assay and survival in serum-free media assays for HeLa cells and A549 cells, respectively, where Dock7 has been knocked down using shRNA. Western blot analysis shows Dock7 protein expression for each cell line. ( C ) Kaplan–Meier survival plot from The Protein Atlas showing a correlation between Dock7 mRNA expression and survival in liver cancer patients. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in ( A - B ) represent means ± SD; ***p < 0.001, and **p < 0.01, and not significant (n.s.)

Article Snippet: Dock7 antibody was purchased from Santa Cruz Biotechnology, PHLPP was purchased from Proteintech, and all other antibodies were obtained from Cell Signaling Technologies.

Techniques: Soft Agar Assay, shRNA, Western Blot, Expressing

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) Either Myc-tagged TSC1, Flag-tagged TSC2, or ( B ) Myc-mTOR were transiently expressed in HEK293T cells and isolated with anti-Myc, or anti-Flag beads. Immunoprecipitated endogenous Dock7 protein was separated and analyzed by Western blot. ( C ) HeLa cells were transiently transfected with either WT Dock7 or Rheb for 48 h after which, the cells were starved overnight and collected for analysis. Heregulin treated (1 nmol/L, 30 mins) and mock-transfected cells were used as positive and negative controls, respectively. Western blots are representative of three separate biological replicates.

Article Snippet: Dock7 antibody was purchased from Santa Cruz Biotechnology, PHLPP was purchased from Proteintech, and all other antibodies were obtained from Cell Signaling Technologies.

Techniques: Isolation, Immunoprecipitation, Western Blot, Transfection

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) Cells were either mock-transfected or transiently transfected with plasmids to overexpress HA-tagged WT Rheb or the active forms of Cdc42 and Rac. Heregulin treatment (1 nmol/L for 1 h) was used as a control to confirm mTORC1 signaling stimulation. ( B ) HA-tagged Cdc42 was overexpressed, and different Rac1 siRNAs were used to knock-down Rac protein. ( C ) HA-tagged Rac1 was overexpressed, and different Cdc42 siRNAs were used to knock-down Cdc42 protein. ( D ) Cells were either mock-transfected or transiently transfected with plasmids to overexpress either V5-tagged Dock7 WT or its GEF-defective mutant (GDM). ( E ) V5-tagged Dock7 was transiently overexpressed, while either Cdc42 or Rac was knocked down using siRNA. All experiments were performed in HeLa cells. After the genetic manipulation specified, cells were serum starved overnight (16–20 h) and collected for Western blot analysis. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states when normalized to endogenous protein levels.

Article Snippet: Dock7 antibody was purchased from Santa Cruz Biotechnology, PHLPP was purchased from Proteintech, and all other antibodies were obtained from Cell Signaling Technologies.

Techniques: Transfection, Control, Knockdown, Mutagenesis, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: CRISPR-Cas9 with Dock7 gene guiding sequence was expressed in cells and antibiotic resistance was applied. Once cells were selected, Dock7 protein expression was determined using western blotting ( A ) and then these cells were used as follows: ( B and C ) Cells were seeded and allowed to grow in full media or ( B ) in soft agar suspension for two weeks ( C ). Colonies formed in soft agar suspension were counted two weeks after seeding. ( D ) Cells were seeded and allowed to recover for a day before changing media to serum-free media for 24 h. Cells were then either collected (Lane 1 and 4), treated with 100 nM insulin for 1 h (Lane 3 and 6), or media was changed to HBBS to remove amino acids for 1 h (Lane 2 and 5) before being collected and used for Western blot analysis. ( E ) Cells were grown in either serum-free or glutamine-free media for two days before they were trypsinized and counted. ( F ) Cells were grown in either complete media or serum-free conditions for 20–24 h and then treated with EdU (10 μm) for 4 h. Cells were then fixed and EdU incorporation was determined. ( G ) Dock7 KO and WT HeLa cells were seeded, starved for 24 h, then stained with TUNEL and cleaved caspase 3 antibody to determine apoptotic index. DAPI stain was used to normalize for cell number. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states when normalized to endogenous protein levels. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in (C and E-G) represent means ± SD; ***p < 0.001, and **p < 0.01, *p < 0.1, and not significant (n.s.)

Article Snippet: Dock7 antibody was purchased from Santa Cruz Biotechnology, PHLPP was purchased from Proteintech, and all other antibodies were obtained from Cell Signaling Technologies.

Techniques: CRISPR, Sequencing, Expressing, Western Blot, Suspension, Staining, TUNEL Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) WT HeLa cells were seeded and culturedfor one day before media was changed to serum-free media containing either vehicle DMSO, Rapamycin (1 nM) or Torin (100 nM). Cells were treated for 20 h, then collected, and lysed for Western blot analysis. ( B ) WT HeLa cells were seeded in soft agar suspension and allowed to recover for a day before treatment began. Cells were then treated with 200 μl of complete media containing either vehicle DMSO, Rapamycin (1 nM), Torin (100 nM), or MK2206 (10 μM) on day 2, and every 3 days subsequently. Colonies were quantified 2 weeks after first drug treatment. ( C ) Either Rictor or Raptor was semi-stably knocked down with shRNA, and then full-length V5-tagged Dock7 protein was overexpressed. After 48 h, cells were starved overnight and collected for Western blot. ( D ) Dock7-V5 containing plasmid was transiently transfected in WT HeLa cells and cells were treated with serum-free media either containing vehicle control (DMSO, 1:100) or Rapamycin (1 nM). Cells were then collected and used for Western blot analysis. ( E ) V5-tagged Dock7 was transiently overexpressed in HeLa cells, and Rheb was knocked down using siRNA the next day. After 48 h, cells were starved overnight and collected for Western blot analysis. ( F ) S6 Kinase was knocked down using siRNA, and cells were seeded in soft agar suspension. Colonies formed were counted two weeks after. S6K protein levels were determined using western blotting. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states normalized to endogenous protein levels. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in ( B and F ) represent means ± SD; ***p < 0.001, and **p < 0.01.

Article Snippet: Dock7 antibody was purchased from Santa Cruz Biotechnology, PHLPP was purchased from Proteintech, and all other antibodies were obtained from Cell Signaling Technologies.

Techniques: Western Blot, Suspension, Stable Transfection, shRNA, Plasmid Preparation, Transfection, Control, Phospho-proteomics

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) Flag-tagged AKT was transiently overexpressed in HEK293T cells semi-stably expressing either V5-DHR1L or V5-DHR2. Cells were then either treated with vehicle control (DMSO, 1:1000) or with MK2206 (10 μM) for 16 h before being collected and lysed. Immunoprecipitation was performed using anti-V5 beads and complexes were resolved on an SDS-PAGE gel for Western analysis. ( B ) HEK293T cells semi-stably expressing either V5-DHR1L or V5-DHR2 were grown in full-serum or serum starved for 16 h, collected, lysed then used for immunoprecipitation and western blot analysis as described in ( A ). Proximity Ligation Assays (PLA) were performed to determine the number of direct interactions between endogenous Dock7 and either ( C ) endogenous AKT or ( D ) phospho-AKT in HeLa cells, respectively. ( E ) Cirspr-Cas9 Dock7 KO and WT HeLa cells were seeded and allowed to recover for 24 h. Media was then changed to either full serum media or serum-free media for 24 h. Serum-starved cells were then treated with either vehicle control DMSO (10 μl), Okadaic Acid (10 nM), or Calyculin A (50 nM) for 1 h before being collected for Western blot analysis. ( F ) Proximity ligation assay was performed on Cirspr-Cas9 Dock7 KO and WT HeLa cells to measure the number of complexes formed between AKT and PHLPP in the presence and absence of Dock7.

Article Snippet: Dock7 antibody was purchased from Santa Cruz Biotechnology, PHLPP was purchased from Proteintech, and all other antibodies were obtained from Cell Signaling Technologies.

Techniques: Stable Transfection, Expressing, Control, Immunoprecipitation, SDS Page, Western Blot, Ligation, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A and B ) Dock7 was semi-stably knocked down using shRNA and either an empty vector or vectors containing the limit DHR domains were expressed using lentiviral system. Cells were then seeded in either ( A ) soft agar suspension and counted two weeks later or ( B ) in 100 mm plates for protein analysis using western blotting. ( C ) Lentiviral transduction system was used to overexpress the specified domains and their mutants in HeLa WT cells. Cells expressing each plasmid were selected with antibiotics for 3–5 days, seeded, and allowed to recover for 24 h. Media was changed to serum-free media and cells were collected after 24 h for Western blot analysis. ( D ) Semi-stable HeLa cells overexpressing either an empty vector, DHR1L, or its C2 mutant were seeded, allowed to recover, and then allowed to grow for four days in serum-free conditions. Cells were then trypsin-treated and counted. ( E - G ) Dock7 was knocked down using shRNA in MDA-MB-231 cells, and then the specified constructs were overexpressed. Cells were then either grown in complete media or starved for 20–24 h. Proximity ligation assays (PLA) were used determine the number of interactions between endogenous AKT or phospho-AKT and each Dock7 construct. Western blots are representative of three separate biological replicates. Survival assays were performed in triplicates. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in (DG) represent means ± SD; ***p < 0.001, and **p < 0.01, *p < 0.1, and not significant (n.s.)

Article Snippet: Dock7 antibody was purchased from Santa Cruz Biotechnology, PHLPP was purchased from Proteintech, and all other antibodies were obtained from Cell Signaling Technologies.

Techniques: Stable Transfection, shRNA, Plasmid Preparation, Suspension, Western Blot, Transduction, Expressing, Mutagenesis, Construct, Ligation

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) In normal tissues, chronic stress will promote cell death. ( B ) When Dock7 is overexpressed in cancer cells and cells are challenged with stress, Dock7 will be able to maintain AKT phosphorylated to inhibit apoptosis and promote survival. Created with BioRender.com

Article Snippet: Dock7 antibody was purchased from Santa Cruz Biotechnology, PHLPP was purchased from Proteintech, and all other antibodies were obtained from Cell Signaling Technologies.

Techniques:

Journal: bioRxiv

Article Title: Deletion of Dock7 Exons 3 and 4 Results in Reduced Trabecular Microarchitecture and a Decrease in Mineralization

doi: 10.64898/2025.12.31.696872

Figure Lengend Snippet: (A) Schematic of the Dock7 wild type (+) and Dock7 exons 3–4 deletion ( Dock7-em2) transgenic allele. Image created with BioRender.com. (B) Predicted translation of the Dock7+ and Dock7-em2 alleles. Image created with BioRender.com. (C) Representative images of Dock7 +/+ , Dock7 +/em2 , and Dock7 em2/em2 mice.

Article Snippet: Dock7 exons 3–4 levels were quantified using a TaqMan gene expression assay containing primers that span across Dock7 exons 3–4 (Thermo Fisher, Mm01259842_g1, 4351372) and TaqMan Fast Advanced Master Mix (Thermo Fisher, 4444556).

Techniques: Transgenic Assay

Journal: bioRxiv

Article Title: Deletion of Dock7 Exons 3 and 4 Results in Reduced Trabecular Microarchitecture and a Decrease in Mineralization

doi: 10.64898/2025.12.31.696872

Figure Lengend Snippet: (A) RNA expression of Dock7 exons 3–4, exons 11–12, and exons 34–35 was analyzed in BMSCs differentiated for 7 days in osteogenic media. Points represent individual replicates for all 3 experiments. (B) Quantitative protein analysis of DOCK7 peptides was performed by mass spectrometry in BMSC isolated from Dock7 +/+ and Dock7 em2/em2 mice. N=2 mice/group. Points represent replicates from all mice. (C) DOCK7 peptides were detected by mass spectrometry and used to quantify total DOCK7 levels. High confidence (green), medium confidence (yellow), and low confidence (red) DOCK7 peptides are indicated. The protein sequence corresponding to DOCK7 exons 3–4, the DHR1 domain, and the DHR2 domain is underlined and labeled.

Article Snippet: Dock7 exons 3–4 levels were quantified using a TaqMan gene expression assay containing primers that span across Dock7 exons 3–4 (Thermo Fisher, Mm01259842_g1, 4351372) and TaqMan Fast Advanced Master Mix (Thermo Fisher, 4444556).

Techniques: RNA Expression, Mass Spectrometry, Isolation, Sequencing, Labeling

Journal: bioRxiv

Article Title: Deletion of Dock7 Exons 3 and 4 Results in Reduced Trabecular Microarchitecture and a Decrease in Mineralization

doi: 10.64898/2025.12.31.696872

Figure Lengend Snippet: Body composition was measured by DXA analysis in male and female Dock7 em2/em2 mice and compared to Dock7 +/+ control mice. Data was analyzed by 2-way ANOVA and Holm-Šídák’s test for post-hoc analysis. N=14–15 mice/group. Points represent each mouse.

Article Snippet: Dock7 exons 3–4 levels were quantified using a TaqMan gene expression assay containing primers that span across Dock7 exons 3–4 (Thermo Fisher, Mm01259842_g1, 4351372) and TaqMan Fast Advanced Master Mix (Thermo Fisher, 4444556).

Techniques: Control

Journal: bioRxiv

Article Title: Deletion of Dock7 Exons 3 and 4 Results in Reduced Trabecular Microarchitecture and a Decrease in Mineralization

doi: 10.64898/2025.12.31.696872

Figure Lengend Snippet: Osteogenic differentiation was assessed in BMSCs that were isolated from Dock7 +/+ and Dock7 em2/em2 mice after 7 days of osteogenic differentiation. (A) Representative image of von Kossa and alkaline phosphatase staining. (B) Expression of the osteoblast-related genes Runx2 , AlpI , and Bglap . Points represent individual replicates for all 3 experiments.

Article Snippet: Dock7 exons 3–4 levels were quantified using a TaqMan gene expression assay containing primers that span across Dock7 exons 3–4 (Thermo Fisher, Mm01259842_g1, 4351372) and TaqMan Fast Advanced Master Mix (Thermo Fisher, 4444556).

Techniques: Isolation, Staining, Expressing

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) The Cancer Genome Atlas (TGCA) expression profile for Dock-C family members, Dock6, Dock7, and Dock8, in triple-negative breast cancer and normal tissue. ( B ) Protein expression of Dock7 in a breast cancer cell-line cohort. ( C ) Dock7 mRNA levels across patients with either receptor-positive (BC) or triple-negative breast cancers (TNBC). ( D and E ) Quantification of colonies formed in soft agar suspension for SKRB3 and MCF7, respectively. (Below) Western blot showing Dock7 protein expression after either control or Dock7 KD for each cell line. ( F ) Dock7 expression profile for MDA-MB-231 cells that were used for either (G) soft agar assay or ( H ) survival in serum-free media for 4 days. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in ( C ), ( D - E ), and ( G - H ) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01

Article Snippet: A solution of 1 μg of CRISPR (Santa Cruz Biotechnology, sc-404461) and DOCK7 HDR (h) (Santa Cruz Biotechnology, sc-404461-HDR) plasmid DNA was added to 150 μl Plasmid Transfection Medium (Santa Cruz Biotechnology, sc-108062) was prepared and mixed.

Techniques: Expressing, Suspension, Western Blot, Control, Soft Agar Assay

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A and B ) Soft agar colony formation assay and survival in serum-free media assays for HeLa cells and A549 cells, respectively, where Dock7 has been knocked down using shRNA. Western blot analysis shows Dock7 protein expression for each cell line. ( C ) Kaplan– Meier survival plot from The Protein Atlas showing a correlation between Dock7 mRNA expression and survival in liver cancer patients. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in ( A - B ) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01, and not significant (n.s.)

Article Snippet: A solution of 1 μg of CRISPR (Santa Cruz Biotechnology, sc-404461) and DOCK7 HDR (h) (Santa Cruz Biotechnology, sc-404461-HDR) plasmid DNA was added to 150 μl Plasmid Transfection Medium (Santa Cruz Biotechnology, sc-108062) was prepared and mixed.

Techniques: Soft Agar Assay, shRNA, Western Blot, Expressing

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) Either Myc-tagged TSC1, Flag-tagged TSC2, or ( B ) Myc-mTOR were transiently expressed in HEK293T cells and isolated with anti-Myc, or anti-Flag beads. Immunoprecipitated endogenous Dock7 protein was separated and analyzed by Western blot. ( C ) HeLa cells were transiently transfected with either WT Dock7 or Rheb for 48 h after which, the cells were starved overnight and collected for analysis. Heregulin treated (1 nmol/L, 30 mins) and mock-transfected cells were used as positive and negative controls, respectively. Western blots are representative of three separate biological replicates.

Article Snippet: A solution of 1 μg of CRISPR (Santa Cruz Biotechnology, sc-404461) and DOCK7 HDR (h) (Santa Cruz Biotechnology, sc-404461-HDR) plasmid DNA was added to 150 μl Plasmid Transfection Medium (Santa Cruz Biotechnology, sc-108062) was prepared and mixed.

Techniques: Isolation, Immunoprecipitation, Western Blot, Transfection

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) Cells were either mock-transfected or transiently transfected with plasmids to overexpress HA-tagged WT Rheb or the active forms of Cdc42 and Rac. Heregulin treatment (1 nmol/L for 1 h) was used as a control to confirm mTORC1 signaling stimulation. ( B ) HA-tagged Cdc42 was overexpressed, and different Rac1 siRNAs were used to knock-down Rac protein. ( C ) HA-tagged Rac1 was overexpressed, and different Cdc42 siRNAs were used to knock-down Cdc42 protein. ( D ) Cells were either mock-transfected or transiently transfected with plasmids to overexpress either V5-tagged Dock7 WT or its GEF-defective mutant (GDM). ( E ) V5-tagged Dock7 was transiently overexpressed, while either Cdc42 or Rac was knocked down using siRNA. All experiments were performed in HeLa cells. After the genetic manipulation specified, cells were serum starved overnight (16-20 h) and collected for Western blot analysis. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states when normalized to endogenous protein levels.

Article Snippet: A solution of 1 μg of CRISPR (Santa Cruz Biotechnology, sc-404461) and DOCK7 HDR (h) (Santa Cruz Biotechnology, sc-404461-HDR) plasmid DNA was added to 150 μl Plasmid Transfection Medium (Santa Cruz Biotechnology, sc-108062) was prepared and mixed.

Techniques: Transfection, Control, Knockdown, Mutagenesis, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: CRISPR-Cas9 with Dock7 gene guiding sequence was expressed in cells and antibiotic resistance was applied. Once cells were selected, Dock7 protein expression was determined using western blotting ( A ) and then these cells were used as follows: ( B and C ) Cells were seeded and allowed to grow in full media or ( B ) in soft agar suspension for two weeks ( C ). Colonies formed in soft agar suspension were counted two weeks after seeding. ( D ) Cells were seeded and allowed to recover for a day before changing media to serum-free media for 24 h. Cells were then either collected (Lane 1 and 4), treated with 100 nM insulin for 1 h (Lane 3 and 6), or media was changed to HBBS to remove amino acids for 1 h (Lane 2 and 5) before being collected and used for Western blot analysis. ( E ) Cells were grown in either serum-free or glutamine-free media for two days before they were trypsinized and counted. ( F ) Cells were grown in either complete media or serum-free conditions for 20-24 h and then treated with EdU (10 μm) for 4 h. Cells were then fixed and EdU incorporation was determined. ( G ) Dock7 KO and WT HeLa cells were seeded, starved for 24 h, then stained with TUNEL and cleaved caspase 3 antibody to determine apoptotic index. DAPI stain was used to normalize for cell number. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states when normalized to endogenous protein levels. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in (C and E-G) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01, ∗p < 0.1, and not significant (n.s.)

Article Snippet: A solution of 1 μg of CRISPR (Santa Cruz Biotechnology, sc-404461) and DOCK7 HDR (h) (Santa Cruz Biotechnology, sc-404461-HDR) plasmid DNA was added to 150 μl Plasmid Transfection Medium (Santa Cruz Biotechnology, sc-108062) was prepared and mixed.

Techniques: CRISPR, Sequencing, Expressing, Western Blot, Suspension, Staining, TUNEL Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) WT HeLa cells were seeded and culturedfor one day before media was changed to serum-free media containing either vehicle DMSO, Rapamycin (1 nM) or Torin (100 nM). Cells were treated for 20 h, then collected, and lysed for Western blot analysis. ( B ) WT HeLa cells were seeded in soft agar suspension and allowed to recover for a day before treatment began. Cells were then treated with 200 μl of complete media containing either vehicle DMSO, Rapamycin (1 nM), Torin (100 nM), or MK2206 (10 μM) on day 2, and every 3 days subsequently. Colonies were quantified 2 weeks after first drug treatment. ( C ) Either Rictor or Raptor was semi-stably knocked down with shRNA, and then full-length V5-tagged Dock7 protein was overexpressed. After 48 h, cells were starved overnight and collected for Western blot. ( D ) Dock7-V5 containing plasmid was transiently transfected in WT HeLa cells and cells were treated with serum-free media either containing vehicle control (DMSO, 1:100) or Rapamycin (1 nM). Cells were then collected and used for Western blot analysis. ( E ) V5-tagged Dock7 was transiently overexpressed in HeLa cells, and Rheb was knocked down using siRNA the next day. After 48 h, cells were starved overnight and collected for Western blot analysis. ( F ) S6 Kinase was knocked down using siRNA, and cells were seeded in soft agar suspension. Colonies formed were counted two weeks after. S6K protein levels were determined using western blotting. Western blots are representative of three separate biological replicates, and numbers under western blots represent fold difference between phosphorylation states normalized to endogenous protein levels. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in ( B and F ) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01.

Article Snippet: A solution of 1 μg of CRISPR (Santa Cruz Biotechnology, sc-404461) and DOCK7 HDR (h) (Santa Cruz Biotechnology, sc-404461-HDR) plasmid DNA was added to 150 μl Plasmid Transfection Medium (Santa Cruz Biotechnology, sc-108062) was prepared and mixed.

Techniques: Western Blot, Suspension, Stable Transfection, shRNA, Plasmid Preparation, Transfection, Control, Phospho-proteomics

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) Flag-tagged AKT was transiently overexpressed in HEK293T cells semi-stably expressing either V5-DHR1L or V5-DHR2. Cells were then either treated with vehicle control (DMSO, 1:1000) or with MK2206 (10 μM) for 16 h before being collected and lysed. Immunoprecipitation was performed using anti-V5 beads and complexes were resolved on an SDS-PAGE gel for Western analysis. ( B ) HEK293T cells semi-stably expressing either V5-DHR1L or V5-DHR2 were grown in full-serum or serum starved for 16 h, collected, lysed then used for immunoprecipitation and western blot analysis as described in ( A ). Proximity Ligation Assays (PLA) were performed to determine the number of direct interactions between endogenous Dock7 and either ( C ) endogenous AKT or ( D ) phospho-AKT in HeLa cells, respectively. ( E ) Cirspr-Cas9 Dock7 KO and WT HeLa cells were seeded and allowed to recover for 24 h. Media was then changed to either full serum media or serum-free media for 24 h. Serum-starved cells were then treated with either vehicle control DMSO (10 µl), Okadaic Acid (10 nM), or Calyculin A (50 nM) for 1 h before being collected for Western blot analysis. ( F ) Proximity ligation assay was performed on Cirspr-Cas9 Dock7 KO and WT HeLa cells to measure the number of complexes formed between AKT and PHLPP in the presence and absence of Dock7.

Article Snippet: A solution of 1 μg of CRISPR (Santa Cruz Biotechnology, sc-404461) and DOCK7 HDR (h) (Santa Cruz Biotechnology, sc-404461-HDR) plasmid DNA was added to 150 μl Plasmid Transfection Medium (Santa Cruz Biotechnology, sc-108062) was prepared and mixed.

Techniques: Stable Transfection, Expressing, Control, Immunoprecipitation, SDS Page, Western Blot, Ligation, Proximity Ligation Assay

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A and B ) Dock7 was semi-stably knocked down using shRNA and either an empty vector or vectors containing the limit DHR domains were expressed using lentiviral system. Cells were then seeded in either ( A ) soft agar suspension and counted two weeks later or ( B ) in 100 mm plates for protein analysis using western blotting. ( C ) Lentiviral transduction system was used to overexpress the specified domains and their mutants in HeLa WT cells. Cells expressing each plasmid were selected with antibiotics for 3-5 days, seeded, and allowed to recover for 24 h. Media was changed to serum-free media and cells were collected after 24 h for Western blot analysis. ( D ) Semi-stable HeLa cells overexpressing either an empty vector, DHR1L, or its C2 mutant were seeded, allowed to recover, and then allowed to grow for four days in serum-free conditions. Cells were then trypsin-treated and counted. ( E - G ) Dock7 was knocked down using shRNA in MDA-MB-231 cells, and then the specified constructs were overexpressed. Cells were then either grown in complete media or starved for 20-24 h. Proximity ligation assays (PLA) were used determine the number of interactions between endogenous AKT or phospho-AKT and each Dock7 construct. Western blots are representative of three separate biological replicates. Survival assays were performed in triplicates. Soft agar assays were performed in triplicates, and colonies formed were quantified using ImageJ. The data shown in (DG) represent means ± SD; ∗∗∗p < 0.001, and ∗∗p < 0.01, ∗p < 0.1, and not significant (n.s.)

Article Snippet: A solution of 1 μg of CRISPR (Santa Cruz Biotechnology, sc-404461) and DOCK7 HDR (h) (Santa Cruz Biotechnology, sc-404461-HDR) plasmid DNA was added to 150 μl Plasmid Transfection Medium (Santa Cruz Biotechnology, sc-108062) was prepared and mixed.

Techniques: Stable Transfection, shRNA, Plasmid Preparation, Suspension, Western Blot, Transduction, Expressing, Mutagenesis, Construct, Ligation

Journal: bioRxiv

Article Title: Dock7 regulates AKT and mTOR/S6K activity required for the transformed phenotypes and survival of cancer cells

doi: 10.1101/2023.01.03.522657

Figure Lengend Snippet: ( A ) In normal tissues, chronic stress will promote cell death. ( B ) When Dock7 is overexpressed in cancer cells and cells are challenged with stress, Dock7 will be able to maintain AKT phosphorylated to inhibit apoptosis and promote survival. Created with BioRender.com

Article Snippet: A solution of 1 μg of CRISPR (Santa Cruz Biotechnology, sc-404461) and DOCK7 HDR (h) (Santa Cruz Biotechnology, sc-404461-HDR) plasmid DNA was added to 150 μl Plasmid Transfection Medium (Santa Cruz Biotechnology, sc-108062) was prepared and mixed.

Techniques:

Journal: Arthritis Research & Therapy

Article Title: The influence of the NOD Nss1 / Idd5 loci on sialadenitis and gene expression in salivary glands of congenic mice

doi: 10.1186/ar2300

Figure Lengend Snippet: Validation of microarray by QPCR

Article Snippet: The 10 assays used for QPCR validation were Ccl19 (Mm00839967_g1), Cd19 (Mm00515420_m1), Egf (Mm00438696_m1), Klk9 ( Egf-bp ) (Mm00658534_mH), Ltb (Mm00434774_g1), Sell (Mm00441291_m1), Zap70 (Mm00494255_m1), Stat1 (Mm00439518_m1), Dock7 (Mm01259863_m1) and Fas (Mm00433237_m1).

Techniques: Biomarker Discovery, Microarray

Journal: Arthritis Research & Therapy

Article Title: The influence of the NOD Nss1 / Idd5 loci on sialadenitis and gene expression in salivary glands of congenic mice

doi: 10.1186/ar2300

Figure Lengend Snippet: Differentially expressed genes in the two congenic intervals

Article Snippet: The 10 assays used for QPCR validation were Ccl19 (Mm00839967_g1), Cd19 (Mm00515420_m1), Egf (Mm00438696_m1), Klk9 ( Egf-bp ) (Mm00658534_mH), Ltb (Mm00434774_g1), Sell (Mm00441291_m1), Zap70 (Mm00494255_m1), Stat1 (Mm00439518_m1), Dock7 (Mm01259863_m1) and Fas (Mm00433237_m1).

Techniques: Membrane, Binding Assay, Glycoproteomics

Journal: Arthritis Research & Therapy

Article Title: The influence of the NOD Nss1 / Idd5 loci on sialadenitis and gene expression in salivary glands of congenic mice

doi: 10.1186/ar2300

Figure Lengend Snippet: Differentially expressed cytokine and chemokine genes

Article Snippet: The 10 assays used for QPCR validation were Ccl19 (Mm00839967_g1), Cd19 (Mm00515420_m1), Egf (Mm00438696_m1), Klk9 ( Egf-bp ) (Mm00658534_mH), Ltb (Mm00434774_g1), Sell (Mm00441291_m1), Zap70 (Mm00494255_m1), Stat1 (Mm00439518_m1), Dock7 (Mm01259863_m1) and Fas (Mm00433237_m1).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Association of the variants and haplotypes in the DOCK 7, PCSK 9 and GALNT 2 genes and the risk of hyperlipidaemia

doi: 10.1111/jcmm.12713

Figure Lengend Snippet: The association between the DOCK7 , PCSK9 and GALNT2 polymorphisms with hypercholesterolaemia

Article Snippet: DOCK7 (gene ID: 85440, MedGen: CN189147, OMIM: 615859) is located on chromosome 1p31.3 (Exon count: 53) and encodes for DOCK7 protein.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Association of the variants and haplotypes in the DOCK 7, PCSK 9 and GALNT 2 genes and the risk of hyperlipidaemia

doi: 10.1111/jcmm.12713

Figure Lengend Snippet: The association between the DOCK7 , PCSK9 and GALNT2 polymorphisms with hypertriglyceridaemia

Article Snippet: DOCK7 (gene ID: 85440, MedGen: CN189147, OMIM: 615859) is located on chromosome 1p31.3 (Exon count: 53) and encodes for DOCK7 protein.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Association of the variants and haplotypes in the DOCK 7, PCSK 9 and GALNT 2 genes and the risk of hyperlipidaemia

doi: 10.1111/jcmm.12713

Figure Lengend Snippet: Association between the genotypes of DOCK7 , PCSK9 and GALNT2 SNPs and serum lipid levels in the hypercholesterolaemic and non‐hypercholesterolaemic individuals

Article Snippet: DOCK7 (gene ID: 85440, MedGen: CN189147, OMIM: 615859) is located on chromosome 1p31.3 (Exon count: 53) and encodes for DOCK7 protein.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Association of the variants and haplotypes in the DOCK 7, PCSK 9 and GALNT 2 genes and the risk of hyperlipidaemia

doi: 10.1111/jcmm.12713

Figure Lengend Snippet: Association between the genotypes of DOCK7 , PCSK9 and GALNT2 SNPs and serum lipid levels in the hypertriglyceridaemic and non‐hypertriglyceridaemic individuals

Article Snippet: DOCK7 (gene ID: 85440, MedGen: CN189147, OMIM: 615859) is located on chromosome 1p31.3 (Exon count: 53) and encodes for DOCK7 protein.

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Association of the variants and haplotypes in the DOCK 7, PCSK 9 and GALNT 2 genes and the risk of hyperlipidaemia

doi: 10.1111/jcmm.12713

Figure Lengend Snippet: The association between the DOCK7, PCSK9 and GALNT2 haplotypes and hypercholesterolaemia/hypertriglyceridaemia

Article Snippet: DOCK7 (gene ID: 85440, MedGen: CN189147, OMIM: 615859) is located on chromosome 1p31.3 (Exon count: 53) and encodes for DOCK7 protein.

Techniques: Control

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

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.

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

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.

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, Incubation, Isolation, Expressing, Transfection, Immunoprecipitation

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.

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

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

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

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.

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: In Vitro, In Vivo, Colony Assay, Injection