Journal: Frontiers in Molecular Neuroscience

Article Title: Major Signaling Pathways in Migrating Neuroblasts

doi: 10.3389/neuro.02.007.2009

Figure Lengend Snippet: In vitro analysis of the cytoskeleton pathway . (A) Cytoskeleton pathway involved in neuroblast migration resulting from the microarray data analysis. (B) Boyden chamber migrational assay: dissected neuroblasts were plated on a porous membrane, were allowed to migrate for 24 hours and Tuj1-positive cells were then counted ( p < 0.001). (C,D) Migration analysis in organotypic cultures obtained from 5HT3 A -EGFP mice. (C1) PI3K inhibitor (LY294002) severely disturbed neuroblast migration. Migration was quantified as the ratio of the EGFP-positive neuroblast containing area surrounding the SVZ between untreated (control) and treated (inhibitor) slices (obtained from the same sagittal level) after 4 days in culture ( n ≥ 5 slices per condition). (C2) Higher magnifications of RMS and SVZ in control and PI3K inhibitor-treated slices. Insets: note that LY294002-treated neuroblasts have short or no neurite compared to control neuroblasts. (C3) Quantification of the LY294002 effect ( n = 5, p < 0.001). (D1) Effect of PKCζ inhibitor. (D2) Directionality of neuroblast migration and, therefore, cell polarization is disturbed after PKCζ inhibitor treatment. Neuroblasts do not migrate in the streams, but migrate in all directions. This is clearly visible in the cortex where there are significantly more neuroblasts in PKCζ inhibitor-treated slices compared to control slices. (D3) Quantification of the PKCζ inhibitor effect ( n = 6, p < 0.005) Abbreviations: Ak – Akt1 inhibitor, C – control, cx – cortex, hp – hippocampus, lv – lateral ventricle, LY – PI3K inhibitor LY294002, P3,4,5 – PIP3,4,5; P4,5 – PIP4,5; P3,4 – PIP3,4; PZ – PKCζ inhibitor, Ra – Rac1 inhibitor, TA – Rho GTPases inhibitor Toxin A of C. difficile , W – PI3K inhibitor wortmannin.

Article Snippet: The following antibodies were used in our analysis: polyclonal rabbit anti-EGFP antibody, 1:10000 (Molecular Probes, USA), mouse anti-III class β-tubulin, Tuj1, 1:500 (Covance, USA), goat anti-CaM I, 1:200 (Santa Cruz, Germany), goat anti-doublecortin, 1:500 (Santa Cruz, Germany), rabbit anti-Akt1, 1:200 (Cell Signaling, USA), mouse anti-Wave1, 1:1000 (Neuromab, USA), rabbit anti-Cdc42, 1:1000 (Santa Cruz, Germany), rabbit anti-PI3K, 1:2000 (Upstate, USA), mouse anti-Rac1 (Cytoskeleton, USA), Alexa 488-conjugated anti-rabbit and anti-mouse secondary antibodies (Molecular Probes, USA), anti-mouse, anti-rabbit and anti-goat Cy3 coupled secondary antibodies (Jackson Immuno Research Laboratories, USA), anti-mouse and anti-rabbit HRP-conjugated secondary antibodies (Vector, USA).

Techniques: In Vitro, Migration, Microarray

Journal: Frontiers in Molecular Neuroscience

Article Title: Major Signaling Pathways in Migrating Neuroblasts

doi: 10.3389/neuro.02.007.2009

Figure Lengend Snippet: In vivo analysis of the cytoskeleton pathway . (A) Position of injection site (arrow) and destination area of migratory fluorescent cells (oval) after 7 or 10 days post-injection. (B) Western blot analysis of transfected HEK cells illustrating successful knockdown of Wave1 , Rac1 , Pik3r1 and Akt1 . (C) Red fluorescent cells in olfactory bulb infected by shRNAScrambled and shRNAAkt1 viruses. Fewer infected cells were found in OB of shRNAAkt1-injected animals. (D) Percentage of infected cells in olfactory bulb relative to total number of infected cells on SVZ-RMS-OB route after injection of shRNA expressing viruses against genes of the cytoskeleton pathway (* p < 0.005). Gene names are under histogram. GCL – granule cell layer, GL – glomerular layer.

Article Snippet: The following antibodies were used in our analysis: polyclonal rabbit anti-EGFP antibody, 1:10000 (Molecular Probes, USA), mouse anti-III class β-tubulin, Tuj1, 1:500 (Covance, USA), goat anti-CaM I, 1:200 (Santa Cruz, Germany), goat anti-doublecortin, 1:500 (Santa Cruz, Germany), rabbit anti-Akt1, 1:200 (Cell Signaling, USA), mouse anti-Wave1, 1:1000 (Neuromab, USA), rabbit anti-Cdc42, 1:1000 (Santa Cruz, Germany), rabbit anti-PI3K, 1:2000 (Upstate, USA), mouse anti-Rac1 (Cytoskeleton, USA), Alexa 488-conjugated anti-rabbit and anti-mouse secondary antibodies (Molecular Probes, USA), anti-mouse, anti-rabbit and anti-goat Cy3 coupled secondary antibodies (Jackson Immuno Research Laboratories, USA), anti-mouse and anti-rabbit HRP-conjugated secondary antibodies (Vector, USA).

Techniques: In Vivo, Injection, Western Blot, Transfection, Infection, shRNA, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Major Signaling Pathways in Migrating Neuroblasts

doi: 10.3389/neuro.02.007.2009

Figure Lengend Snippet: Phenotypes of animals infected into aSVZ/pRMS by AAV viruses expressing gene-specific shRNA and red fluorescent protein .

Article Snippet: The following antibodies were used in our analysis: polyclonal rabbit anti-EGFP antibody, 1:10000 (Molecular Probes, USA), mouse anti-III class β-tubulin, Tuj1, 1:500 (Covance, USA), goat anti-CaM I, 1:200 (Santa Cruz, Germany), goat anti-doublecortin, 1:500 (Santa Cruz, Germany), rabbit anti-Akt1, 1:200 (Cell Signaling, USA), mouse anti-Wave1, 1:1000 (Neuromab, USA), rabbit anti-Cdc42, 1:1000 (Santa Cruz, Germany), rabbit anti-PI3K, 1:2000 (Upstate, USA), mouse anti-Rac1 (Cytoskeleton, USA), Alexa 488-conjugated anti-rabbit and anti-mouse secondary antibodies (Molecular Probes, USA), anti-mouse, anti-rabbit and anti-goat Cy3 coupled secondary antibodies (Jackson Immuno Research Laboratories, USA), anti-mouse and anti-rabbit HRP-conjugated secondary antibodies (Vector, USA).

Techniques: Infection, Expressing, shRNA

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 1. Loss of NF1 reduces RAC1-driven melanoblast migration. A. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 9 h and 12 h using either WT or NF1+/−melanoblasts (MB) in the presence of a RAC1 activator (CN04). B. RAC1 activity was measured by G-lisa in WT and NF1+/−melanoblasts (MB). C. Scratch-like migration assay after 3 h, 6 h, 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1). D. Expression status of NF1 and expression of phosphorylated and non-phosphorylated ERK and AKT in NF1+/−melanoblasts by western blot. α-actinin was used as a loading control. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody. E. Scratch-like migration assay representing the percentage of cell coverage after 9 h and 12 h in NF1+/−melanoblasts 48 h-post transfection with either a scramble siRNA (SCR) or with an NF1-specific siRNA (siNF1) and in the presence or absence of a RAC1 activator (CN04). *: SCR vs. siNF1, #: -CN04 vs. +CN04. F. GTP-RAC1 pulldown and total lysates were blotted with α-RAC1 antibody in the presence or absence of a RAC1 activator (CN04). **P < 0.01, *P < 0.05, ns: not significant (unpaired Student's t-test). All error bars represent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Activity Assay, Transfection, Expressing, Western Blot, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 2. Loss of NF1 increases melanoma migration and is associated with increased PREX1 expression. A. NF1 mRNA expression under NF1 silencing with two siRNAs (NF1.6 and NF1.11) in SK-mel-23, Mel501, and SK-mel-103 melanoma cell lines. B. PREX1 mRNA expression under NF1 silencing with two siRNAs in SK-mel-23, Mel501, and SK- mel-103 cell lines. C. Scratch-like migration assay representing the percentage of cell coverage after 6 h, 12 h and 24 h under NF1 silencing in SK-mel-23, Mel501, and SK-mel- 103 cell lines. D. Scratch-like migration assay as in C, after additional transfection with siRNA control (scramble) or with PREX1 siRNA (siPREX1). E. Scratch-like migration assay as in C. in the absence (control) or presence (RAC1 inhibitor) of a RAC1 inhibitor. ***P < 0.001, **P < 0.01, *P < 0.05 (unpaired Student's t-test). All error bars rep- resent the SEM of at least three independent experiments.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Migration, Expressing, Transfection, Control

Journal: Translational oncology

Article Title: NF1-RAC1 axis regulates migration of the melanocytic lineage.

doi: 10.1016/j.tranon.2020.100858

Figure Lengend Snippet: Fig. 4. PREX is upregulated in low NF1 expressing melanoma metastases. A. Representative microphotographs of Tissue Microarray (TMA) containing primary and metastatic melanoma samples analysed by immunohistochemistry using a specific antibody against NF1, RAC1 and PREX1. Bar, 100 μm. B. Scoring of the immunohistochemistry staining was performed according to our previously described protocol [24]. Duplicates of valid punch samples are represented for each condition. Significance was tested using two-tailed t-test with *P < 0.05 and ns: not significant.

Article Snippet: The amount of activated RAC1 was determined by western blot using a RAC1 specific antibody (Cytoskeleton inc. Cat. # BK035).

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Two Tailed Test

Journal: Oncogene

Article Title: Microphthalmia-Associated Transcription Factor Suppresses Invasion by Reducing Intracellular GTP Pools

doi: 10.1038/onc.2016.178

Figure Lengend Snippet: (A) SK-Mel-28 and 501Mel cells transduced with control vector, or two different shRNAs to MITF were collected as described in Material and Methods and tested in the GTPase pull-down assay followed by immunoblotting with RAC1 antibodies (representative images and quantification of at least 2 independent experiments are shown). (B) SK-Mel-28 cells as in (A) were collected as described in Material and Methods and tested in the GTPase pull-down assay followed by immunoblotting with RHO-A and RHO-C antibodies (representative images and quantification of at least 2 independent experiments are shown). (C) SK-Mel-28 cells as in (A) were treated with the RAC1 inhibitor NSC23766 (25µM) and tested for invasion through matrigel. The data represents the average −/+ SEM of at least two independent. * p <0.05; ** p <0.01 compared to control; # p <0.05 compared to untreated. Statistics performed by Student t-Test.

Article Snippet: The following antibodies were used: mouse monoclonal to MITF (clone D5, Cat# M3621, Dako, Carpinteria, CA); mouse monoclonal to GAPDH (Cat. # AM4300, Ambion, Austin,TX); rabbit polyclonal to GMPR (Cat # SAB1101144 Sigma-Aldrich, St. Louis, MO); rabbit polyclonal to RAC1 (Cell Signaling, Cat# 2465); rabbit monoclonal to RHO-A (Cell Signaling, Cat# 2117); and rabbit monoclonal to RHO-C (Cell Signaling, Cat# 3430).

Techniques: Transduction, Control, Plasmid Preparation, Pull Down Assay, Western Blot

Journal: Oncogene

Article Title: Microphthalmia-Associated Transcription Factor Suppresses Invasion by Reducing Intracellular GTP Pools

doi: 10.1038/onc.2016.178

Figure Lengend Snippet: (A) SK-Mel-28 and A375 human melanoma cells were transduced with an empty vector or an overexpression vector encoding for MITF and probed in invasion assay; cells were supplemented or not with 100µM guanosine for 24hrs prior to and throughout the invasion assay. (B) Cells as in (A) were collected as described in Material and Methods and collected for nucleotides quantification by HPLC. (C) Cells as in (A) were collected as described in Material and Methods and tested in the GTPase pull-down assay followed by immunoblotting with RAC1 antibodies (representative images and quantification of at least two independent experiments are shown). (D) SK-Mel-28 cells as in (A) were collected as described in Material and Methods and tested in the GTPase pull-down assay followed by immunoblotting with RHO-A and RHO-C antibodies (representative images and quantification of at least two independent experiments are shown). The data represents the average −/+ SEM of at least two independent experiments performed in duplicates * p <0.05; ** p <0.001 compared to control; # p <0.05 compared to MITF-overexpressing control. Statistics performed by Student t-Test.

Article Snippet: The following antibodies were used: mouse monoclonal to MITF (clone D5, Cat# M3621, Dako, Carpinteria, CA); mouse monoclonal to GAPDH (Cat. # AM4300, Ambion, Austin,TX); rabbit polyclonal to GMPR (Cat # SAB1101144 Sigma-Aldrich, St. Louis, MO); rabbit polyclonal to RAC1 (Cell Signaling, Cat# 2465); rabbit monoclonal to RHO-A (Cell Signaling, Cat# 2117); and rabbit monoclonal to RHO-C (Cell Signaling, Cat# 3430).

Techniques: Transduction, Plasmid Preparation, Over Expression, Invasion Assay, Pull Down Assay, Western Blot, Control

Journal: Journal of Functional Foods

Article Title: Oxyresveratrol from mulberry (Morus alba L.) ameliorates post-inflammatory hyperpigmentation in vitro by anti-melanogenesis, inhibiting melanosome transfer, and providing photoprotection

doi: 10.1016/j.jff.2024.106557

Figure Lengend Snippet: Fig. 4. The inhibitory effects of OXY, RSV, and GLA on melanin transport in human melanocytes. (a) 15.67 μg/mL OXY, RSV, and GLA were cultured in human melanocytes for 48 h. The mRNA levels of melanin transport genes RAC1, RAB27A, and CDC42 were determined by qRT-PCR. (b) The protein expression level of melanin transport genes RAC1, RAB27A, and CDC42 was determined by western blotting using GAPDH as a loading control. Values are means ± SD of three in dependent experiments run in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared to the control.

Article Snippet: Primary antibodies against CDC42 (10155–1- AP), RAB27A (17817–1-AP), RAC1 (24072–1-AP), and GAPDH (60004–1-Ig), beta-actin polyclonal antibody (β-actin, 20536–1-AP) were purchased from ProteinTech (Wuhan, China).

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Western Blot, Control

Journal: Journal of Functional Foods

Article Title: Oxyresveratrol from mulberry (Morus alba L.) ameliorates post-inflammatory hyperpigmentation in vitro by anti-melanogenesis, inhibiting melanosome transfer, and providing photoprotection

doi: 10.1016/j.jff.2024.106557

Figure Lengend Snippet: Fig. 5. Interaction of OXY with RAB27A, RAC1, and CDC42. The molecular docking results between OXY and RAB27A (a), RAC1 (b), and CDC42 (c) were visualized by PyMOL. The protein models of RAB27A (ID: 3BC1), RAC1 (ID: 3TH5), and CDC42 (ID: 1E0A) were downloaded from the RCSB Protein Data Bank. The ligand, metal ion and protein were labeled in red, yellow, and blue, respectively. The hydrogen bonds between the ligand and protein were repre sented by dotted yellow lines. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Primary antibodies against CDC42 (10155–1- AP), RAB27A (17817–1-AP), RAC1 (24072–1-AP), and GAPDH (60004–1-Ig), beta-actin polyclonal antibody (β-actin, 20536–1-AP) were purchased from ProteinTech (Wuhan, China).

Techniques: Labeling

Journal: Journal of Biological Chemistry

Article Title: AKAP-Lbc Anchors Protein Kinase A and Nucleates Gα12-selective Rho-mediated Stress Fiber Formation

doi: 10.1074/jbc.m106629200

Figure Lengend Snippet: FIG. 4. AKAP-Lbc associates with Rho in vivo. A, AKAP-Lbc was immunoprecipitated from transfected HEK293 cells. Immunoprecipitates were used in an in vitro interaction assay to pull down purified GST-RhoA, GST-Rac1, or GST-Cdc42 preloaded with either GDP or GTPS or nucleotide-depleted (no nucl.). RhoA (lanes 4–6), Rac1 (lanes 7–9), and Cdc42 (lanes 10–12) were detected with anti-GST antibody. B, lysates from untransfected HeLa-S3 cells were subjected to immunoprecipitation with preimmune serum (Pre-i) or affinity-purified anti-AKAP-Lbc antibody. Immunoprecipitates as well as HeLa-S3 cell extracts (Ex.) were separated by SDS-PAGE (4–15%) and electrotransferred to nitrocellulose. RhoA, Rac1, and Cdc42 were detected by immunoblot using monoclonal antibodies against RhoA (lanes 1–3; upper panel), Rac1 (lanes 4–6), or Cdc42 (lanes 7–9), respectively. AKAP-Lbc was detected with an affinity-purified anti-AKAP-Lbc polyclonal antibody (lower panel). C, HEK293 cells were transfected with AKAP-Lbc in combination with either wild type (WT), constitutively active (V14), or dominant negative (N19) RhoA. AKAP-Lbc immunoprecipitates as well as cell extracts (Ex.) were separated by SDS-PAGE (4–15%) and electrotransferred to nitrocellulose. AKAP-Lbc and RhoA were detected by immunoblot using an affinity-purified anti-AKAP-Lbc antibody (lower panel) and a monoclonal anti-RhoA antibody (upper panel). D, HEK293 cells were transfected with the AKAP-Lbc Y2153F mutant in combination with the cDNA encoding wild-type RhoA. Lysates were subjected to immunoprecipitation with preimmune serum (Pre-i) or anti-AKAP-Lbc antibody. RhoA and AKAP-Lbc were detected by immunoblot using a monoclonal anti-RhoA antibody (upper panel) or anti-AKAP-Lbc (lower panel). All of the results are representative of three independent experiments.

Article Snippet: The following affinity-purified primary antibodies were used for immunoblotting: rabbit polyclonal antibody to RhoA (200 g/ml, 1:250 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse monoclonal antibodies to RhoA, Rac1, or Cdc42 (200 g/ml, 1:250 dilution; Santa Cruz Biotechnology); mouse monoclonal antibody to PKA catalytic subunit (clone 5B, 1:1000 dilution; Transduction Laboratories); mouse monoclonal to PKA type II regulatory subunit (1:250 dilution; Transduction Laboratories); mouse monoclonal antibody to the GST tag (1:500 dilution; Santa Cruz Biotechnology); mouse monoclonal antibody to the FLAG tag (1:2000 dilution; Sigma); rabbit polyclonal antibody to the GFP tag (1:100 dilution; Invitrogen); rabbit polyclonal antibody to G 12 (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G 13 (1:250 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G q (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G 11 (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G i2 (1:500 dilution; Santa Cruz Biotechnology); and rabbit polyclonal antibody to G s (1:500 dilution; Santa Cruz Biotechnology).

Techniques: In Vivo, Immunoprecipitation, Transfection, In Vitro, Purification, Affinity Purification, SDS Page, Western Blot, Bioprocessing, Dominant Negative Mutation, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: AKAP-Lbc Anchors Protein Kinase A and Nucleates Gα12-selective Rho-mediated Stress Fiber Formation

doi: 10.1074/jbc.m106629200

Figure Lengend Snippet: FIG. 6. AKAP-Lbc is a Rho-specific guanine nucleotide exchange factor. A, model depicting the guanine nucleotide exchange reaction mediated by AKAP- Lbc. B, time course for the binding of [35S]GTPS to purified RhoA. Purified RhoA was preloaded with GDP and then added to reaction incubations containing [35S]GTPS together with aliquots from HEK293 cell lysates overexpressing AKAP-Lbc (solid circles) or the AKAP-Lbc Y2153F mutant (open circles). C, time course for the dissociation of [3H]GDP from purified RhoA. Purified RhoA was preloaded with [3H]GDP and then added to reaction incubations containing 1 mM GTP together with aliquots from HEK293 cell lysates overexpressing AKAP-Lbc (solid circles) or the AKAP-Lbc Y2153F mutant (open circles). D, effect of AKAP- Lbc on [3H]GDP dissociation from either RhoA Rac1 or Cdc42. Each GTP-binding protein was preloaded with [3H]GDP and then mixed in reaction buffer with ex- tracts overexpressing AKAP-Lbc (solid bars) or the AKAP-Lbc Tyr2153 mutant (gray bars) for 20 min before termination of reactions. Results are expressed as mean S.E. of three independent experiments.

Article Snippet: The following affinity-purified primary antibodies were used for immunoblotting: rabbit polyclonal antibody to RhoA (200 g/ml, 1:250 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); mouse monoclonal antibodies to RhoA, Rac1, or Cdc42 (200 g/ml, 1:250 dilution; Santa Cruz Biotechnology); mouse monoclonal antibody to PKA catalytic subunit (clone 5B, 1:1000 dilution; Transduction Laboratories); mouse monoclonal to PKA type II regulatory subunit (1:250 dilution; Transduction Laboratories); mouse monoclonal antibody to the GST tag (1:500 dilution; Santa Cruz Biotechnology); mouse monoclonal antibody to the FLAG tag (1:2000 dilution; Sigma); rabbit polyclonal antibody to the GFP tag (1:100 dilution; Invitrogen); rabbit polyclonal antibody to G 12 (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G 13 (1:250 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G q (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G 11 (1:500 dilution; Santa Cruz Biotechnology); rabbit polyclonal antibody to G i2 (1:500 dilution; Santa Cruz Biotechnology); and rabbit polyclonal antibody to G s (1:500 dilution; Santa Cruz Biotechnology).

Techniques: Binding Assay, Purification, Mutagenesis