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  • 90
    Thermo Fisher flna
    Flna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore anti flna
    Functional analyses of the exon 40-skipped <t>FLNA.</t> ( a ) <t>GST</t> pull-down assay of FLNA with GST-fused Integrin β7 (GST-ITG). WT, wild-type FLNA; 4del, truncated FLNA and skip, FLNA internally lacking 41 AAs. Representative results of three independent
    Anti Flna, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    3M Co flna exonn
    Cell Type-specific Alternative Splicing of Filamin A in Cerebral Cortical Development and Human PVNH A) RNA-Seq reads around <t>Flna</t> <t>exonN</t> showing its higher inclusion in adult cerebral cortex, cerebellum and E14.5 CP, in comparison to E14.5 VZ and other non-neural adult tissues. B) Flna exonN has an in-frame stop codon. C) ExonN inclusion is predicted to truncate Flna protein. D) Blocking of NMD by cycloheximide (CHX) in primary hippocampal neurons cultured in vitro for 1 day increases inclusion of exonN. Data are represented as mean +/− SEM. E) RNA-Seq reads showing the inclusion of exonN and retention of its upstream intron in fetal human CP. F) Filamin A exonN is conserved across representative placental mammals. G) T1 brain MRI shows PVNH (white arrows) in affected individuals PH-01 (male) and PH-04 (female), compared to a healthy control and an unrelated individual with a FLNA null mutation. H) Pedigree showing inheritance of PVNH and the rare c.1429 +182 G > A mutation. I) Sanger sequencing traces showing the rare FLNA variant c.1429+182 G > A. J) A cartoon illustrating the FLNA mini-gene construct and primers used for RT-PCR. N-terminal HA and Flag tags were fused in-frame with exon 8. K) RT-PCR using primer F and R1 (inside exonN) detecting the abnormal exonN inclusion in blood samples of affected individuals. L) RT-PCR (primer pair F-R2) results on FLNA mini-genes expressed in Neuro2a cells, showing that the c.1429+182 G > A mutation caused > 40% of FLNA transcripts to include exonN. M) Western blot of transfected Neuro2a cells detecting the WT mini-FLNA (green, arrow) and the truncated form (arrow head). Red, anti-Gapdh. N) RT-PCR (primer pair F-R2) results of E14.5 mouse brains electroporated FLNA mini-genes on E13.5, showing that the G > A mutation caused abnormal inclusion of exonN. .
    Flna Exonn, supplied by 3M Co, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    flna  (Abcam)
    90
    Abcam flna
    Validation of the proteomics results by qPCR and Western blotting. (A) Results of qPCR for the mRNA levels of <t>SDHA,</t> SDHB, RhoGDIα, and <t>FLNA.</t> (B) Results of Western blotting for SDHA, SDHB, RhoGDIα, and FLNA. (C) Densitometric analysis of relative protein expressions, GAPDH was used as internal control. n = 4 per group. ∗ P
    Flna, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Santa Cruz Biotechnology anti flna
    <t>Cyclin</t> D1 and <t>FLNa</t> co-localize in cell ruffles. MDA-MB-231 cells were stained for cyclin D1 (Green), FLNa (Red) and DNA (blue). Co-localization of cyclin D1 and FLNa is indicated by the yellow color in the merged picture. The scale bar is 10 µm.
    Anti Flna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore mouse anti flna
    Co-purification of <t>FLNA</t> and <t>integrin</t> β1 and the effects of down-regulation of PrP on integrin β1 and FLNA association. A , left panels , immunoblots ( IM ) show that PrP co-purifies ( IP ) with FLNA, and integrin β1 co-purifies with
    Mouse Anti Flna, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Epitomics flna
    <t>FlnA</t> physically interacts with <t>RhoA</t> and regulates RhoA activation to direct neural progenitor proliferation and differentiation. ( A ) Rho GTPase pull-down assays shows that only active (ACT) or WT-RhoA but not Cdc42 or Rac1 pulls down FlnA by Western blot (left panel) in Neuro2A cells. Conversely, RhoA is co-immunoprecipitated by FlnA C-terminal whereas WT or active Cdc42 and Rac1 did not show any significant binding interaction on Western blot (middle panel). Lastly, full-length FLNA can be co-immunoprecipitated by Flag-RhoA, whereas Flag-Rac1 and Cdc42 cannot (right panel). SUP = supernatant, LAD = molecular weight ladder. Control reflects empty FLAG vector lacking the Rho GTPase constructs followed by pull-down with anti-FLAG antibody and probing for FlnA. ( B ) Fluorescent photomicrographs of cerebral cortex demonstrate FlnA overlapping expression with RhoA in E14 brain cortex, especially, along the apical lining by co-immunostaining and at focal adhesion sites (arrows) of neuroblastoma cells. ( C ) FlnA loss impairs RhoA activation. At baseline, activated RhoA levels are diminished in null FlnA mice. Following laminin activation, RhoA GTPase dramatically increases in the FlnA y/− neural progenitors by 30 min but also declines more precipitously than WT progenitors at 60 min. WT and FlnA y/− neural progenitors are plated on uncoated (uncoated con) or laminin-coated dishes and RhoA activation is assessed at varied time points (30 and 60 min post plating). Upper band corresponds to activated RhoA, and lower band reflects total RhoA (T-RhoA) for loading control. The graph at the right represents mean ± SD values from 5 independent Western blot assays. ( D ) Fluorescent photomicrographs of E14 cerebral cortices 24 h after in utero electroporation of control GFP or dominant negative RhoA (DN-RhoA). Fewer neural cells expressing the DN-RhoA construct reach the cortical plate from the ventricular zone within this time frame, suggesting either impaired migration or diminished neurogenesis. The electroporations specifically target mitotic, proliferating progenitors at the ventricular lining. Progenitors which are therefore GFP + but Ki67- have exited the cell cycle. Higher magnification images and statistical analyses are seen to the right. Co-immunostaining was performed with anti-Ki-67 (marker of cells that remain in the cell cycle) and GFP (marker that initially captures proliferating progenitors) antibodies. The number of GFP + cells that are situated above the ventricular zone is increased in the electroporated GFP control mice (brackets in higher magnification), consistent with increased differentiation. ( E ) The GFP + ,Ki-67 + cells and total GFP + cells from same position-matched area of cortices were quantified. The statistical analysis ( n > 4) show that the ratio of the GFP + , Ki-67 + cells in total GFP + cells is increased approximately 25% in the DN-RhoA electroporation construct versus control. These observations mirror the FlnA loss of function cortical phenotypes and reinforce the likely functional interaction between FlnA and RhoA. Scale bar = 50 μm. ( F ) Fluorescent photomicrographs of GFP (fluorescein) expressing neural cells following electroporation of control GFP or DN-RhoA into lateral ventricles of E13.5 cerebral cortex and staining for early neuronal markers (Tuj1). Acute RhoA inhibition leads to reduction of early born neurons within 72 h, similar to that seen with loss of FlnA. ( G ) Quantification of neurogenic fates is graphically summarized. * = P
    Flna, supplied by Epitomics, used in various techniques. Bioz Stars score: 90/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc anti flna
    <t>Cyclin</t> D1 and <t>FLNa</t> co-localize in cell ruffles. MDA-MB-231 cells were stained for cyclin D1 (Green), FLNa (Red) and DNA (blue). Co-localization of cyclin D1 and FLNa is indicated by the yellow color in the merged picture. The scale bar is 10 µm.
    Anti Flna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Epitomics rabbit anti flna monoclonal antibody
    <t>FlnA</t> regulates FlnB expression levels in chondrocytes and vice versa ( A ) Western blot and <t>immunostaining</t> analysis of FlnA and FlnB expression levels in FlnB knockdown and FlnA knockdown ATDC5 cells, respectively. FlnA protein levels are increased with FlnB inhibition. Conversely, FlnB levels are increased with FlnA inhibition. Levels are graphically quantified to the right. ( B ) Fluorescent photomicrograph demonstrates increased FlnA immunostaining (rhodamine) in stably transfected ATDC5 cells, lacking FlnB (FlnBsh), relative to control. Occasional control cells do show increased FlnA expression, with the vast majority exhibiting lower levels of staining. ( C ) Fluorescent photomicrograph demonstrates increased FlnA immunostaining (rhodamine) in stably transfected ATDC5 cells, lacking FlnB (FlnBsh), relative to control. ( D ) Fluorescent photomicrographs of the growth plate from the radius of newborn FlnB -/- mice. Immnostaining shows FlnA (rhodamine) expression is decreased in the hypertrophic zone (double arrows) but the increased in the proliferative zone (single arrow). ( E ) Similarly, FlnB expression (rhodamine) is decreased in the hypertrophic zone but increased in the proliferative zone of FlnA -/- mice. Scale bars in B and C =100 μm, D and E = 200 μm. Quantification is based on n ≥ 3 tissues sections/western blots per experiment and n > 50 cells.
    Rabbit Anti Flna Monoclonal Antibody, supplied by Epitomics, used in various techniques. Bioz Stars score: 86/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher copy number variation flna mm00516104 cn
    <t>FlnA</t> regulates FlnB expression levels in chondrocytes and vice versa ( A ) Western blot and <t>immunostaining</t> analysis of FlnA and FlnB expression levels in FlnB knockdown and FlnA knockdown ATDC5 cells, respectively. FlnA protein levels are increased with FlnB inhibition. Conversely, FlnB levels are increased with FlnA inhibition. Levels are graphically quantified to the right. ( B ) Fluorescent photomicrograph demonstrates increased FlnA immunostaining (rhodamine) in stably transfected ATDC5 cells, lacking FlnB (FlnBsh), relative to control. Occasional control cells do show increased FlnA expression, with the vast majority exhibiting lower levels of staining. ( C ) Fluorescent photomicrograph demonstrates increased FlnA immunostaining (rhodamine) in stably transfected ATDC5 cells, lacking FlnB (FlnBsh), relative to control. ( D ) Fluorescent photomicrographs of the growth plate from the radius of newborn FlnB -/- mice. Immnostaining shows FlnA (rhodamine) expression is decreased in the hypertrophic zone (double arrows) but the increased in the proliferative zone (single arrow). ( E ) Similarly, FlnB expression (rhodamine) is decreased in the hypertrophic zone but increased in the proliferative zone of FlnA -/- mice. Scale bars in B and C =100 μm, D and E = 200 μm. Quantification is based on n ≥ 3 tissues sections/western blots per experiment and n > 50 cells.
    Copy Number Variation Flna Mm00516104 Cn, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam anti flna rabbit
    Ndr2-deficiency in murine CD4 + T cells attenuates TCR-induced <t>FLNa</t> phosphorylation at <t>S2152,</t> T-cell adhesion and LFA-1-dependent upregulation of CD69 in vitro . (A) Purified splenic wild type (WT) and Ndr2 −/− CD4 + T cells were left untreated or stimulated with anti-CD3 antibodies for the indicated time points. Lysates were prepared and analyzed by Western blotting using the indicated antibodies. Densitrometric quantification of FLNa phosphorylation at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 3). (B) Purified splenic WT and Ndr2 −/− CD4 + T cells were left untreated (non) or stimulated with anti-CD3 antibodies (CD3) and subsequently analyzed for their ability to bind plate-bound Fc-ICAM-1. Adherent cells were counted and calculated as percentage of input ( n = 3). (C) Purified splenic CD4 + T cells from WT and Ndr2 mice were cultured with plate-bound anti-CD3 antibodies (CD3) in the absence or presence of Fc-ICAM-1 (ICAM-1) with or without blocking LFA-1 antibodies (LFA-1) for 12 h. The upregulation of the activation marker CD69 of unstimulated (0 h) or activated T cells (12 h) were assessed by flow cytometry to determine the mean fluorescence intensity (MFI) ( n = 3). (mean ± SEM; * p ≤ 0.05, ** p ≤ 0.01).
    Anti Flna Rabbit, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    OriGene recombinant flna
    Model representing the in vivo <t>S6K-mediated</t> phosphorylation of <t>FLNA,</t> which contributes to S6K-dependent cell shape changes. Cell shape changes occur in vivo during both inflammatory processes and cancer progression and the present study supports an active
    Recombinant Flna, supplied by OriGene, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology goat anti flna
    CCR2B localizes at the tips of actin filaments. CCR2B-A7 ( A ) and CCR2B-M2 ( B ) cells were fixed and incubated with antibodies and analyzed as described before. ( C ) CCR2B-A7 cells were fixed, permeabilized and stained with anti-FLAG Cy3 and <t>FITC-phalloidin.</t> Images are from one single layer of the Z stacks. The colocalization between CCR2B (red) and actin (green) was analyzed using Imaris colocalization software and is shown in white. Bars,10 µm. ( D ) CCR2B-A7 cells were fixed, permeabilized and stained with anti-FLAG Cy3, FITC-phalloidin, mouse <t>anti-FLNa</t> followed by goat anti-mouse Alexa633. Images show pseudo-colors for visualization purposes. Colocalization 1 (β-actin vs. CCR2B) and Colocalization 2 (FLNa vs. CCR2B) were analyzed using Imaris colocalization software and are shown in white. Dotted lines in zoom 1 and zoom 2 mark the direction of the stress fibers. Bars, 10 µm. Experiments were done in duplicates and repeated three times.
    Goat Anti Flna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Thermo Fisher human flna
    CCR2B localizes at the tips of actin filaments. CCR2B-A7 ( A ) and CCR2B-M2 ( B ) cells were fixed and incubated with antibodies and analyzed as described before. ( C ) CCR2B-A7 cells were fixed, permeabilized and stained with anti-FLAG Cy3 and <t>FITC-phalloidin.</t> Images are from one single layer of the Z stacks. The colocalization between CCR2B (red) and actin (green) was analyzed using Imaris colocalization software and is shown in white. Bars,10 µm. ( D ) CCR2B-A7 cells were fixed, permeabilized and stained with anti-FLAG Cy3, FITC-phalloidin, mouse <t>anti-FLNa</t> followed by goat anti-mouse Alexa633. Images show pseudo-colors for visualization purposes. Colocalization 1 (β-actin vs. CCR2B) and Colocalization 2 (FLNa vs. CCR2B) were analyzed using Imaris colocalization software and are shown in white. Dotted lines in zoom 1 and zoom 2 mark the direction of the stress fibers. Bars, 10 µm. Experiments were done in duplicates and repeated three times.
    Human Flna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bethyl rabbit anti filamin a antibody affinity purified
    CCR2B localizes at the tips of actin filaments. CCR2B-A7 ( A ) and CCR2B-M2 ( B ) cells were fixed and incubated with antibodies and analyzed as described before. ( C ) CCR2B-A7 cells were fixed, permeabilized and stained with anti-FLAG Cy3 and <t>FITC-phalloidin.</t> Images are from one single layer of the Z stacks. The colocalization between CCR2B (red) and actin (green) was analyzed using Imaris colocalization software and is shown in white. Bars,10 µm. ( D ) CCR2B-A7 cells were fixed, permeabilized and stained with anti-FLAG Cy3, FITC-phalloidin, mouse <t>anti-FLNa</t> followed by goat anti-mouse Alexa633. Images show pseudo-colors for visualization purposes. Colocalization 1 (β-actin vs. CCR2B) and Colocalization 2 (FLNa vs. CCR2B) were analyzed using Imaris colocalization software and are shown in white. Dotted lines in zoom 1 and zoom 2 mark the direction of the stress fibers. Bars, 10 µm. Experiments were done in duplicates and repeated three times.
    Rabbit Anti Filamin A Antibody Affinity Purified, supplied by Bethyl, used in various techniques. Bioz Stars score: 93/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Epitomics rabbit anti flna
    Expression of <t>FLNA</t> rescues the radial migration defect of Rcan1 knockdown neurons. A , Western blot analysis showing that expression of the FLNA-EGFP construct increased FLNA protein in cultured cortical neurons. The 0 DIV cortical neurons were electroporated with pSuper-scramble (NC) or Rcan1 #1 shRNA (#1) in combination with empty vector (Control) or FLNA-EGFP (FLNA). Cells were lysed 4 d (4 DIV) after electroporation for Western blot. <t>β-Actin</t> was used as a loading control. B , Quantification of the data obtained as in A . Data are mean ± SEM; n = 3. C , D , Representative images ( C ) and statistics results ( D ) showing that overexpression of FLNA did not affect neuronal migration at P7. IUE was performed using pCAG-EGFP combined with pCMV-EGFP-N1 empty vector (Control) or pcDNA3-FLNA-EGFP (FLNA). Scale bar, 300 μm. Quantification (mean ± SEM) of EGFP-positive cell distribution in the cortices of P7. n = 4 for control; n = 5 for FLNA; t test. E , F , Representative images ( E ) and statistics results ( F ) showing that FLNA could remarkably rescue the migration defect and neuronal clustering caused by Rcan1 knockdown. Rescue experiment was performed by using Rcan1 #1 shRNA combined with pCMV-EGFP-N1 empty vector (Control) or pcDNA3-FLNA-EGFP (FLNA). Scale bars: E , Top, 300 μm; E , Bottom, 50 μm. Quantification (mean ± SEM) of EGFP-positive cell distribution in the cortices of P7. n = 9 for control; n = 4 for FLNA. * p
    Rabbit Anti Flna, supplied by Epitomics, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Santa Cruz Biotechnology mouse anti flna
    Loss of <t>FlnA</t> and Fmn2 leads to a defect in cell proliferation along the ventral thoracic wall. A. Fluorescent photomicrographs of immunostaining performed on transverse sections of E13.5 mouse thorax for proliferation markers. Ki-67 (a marker for cells in the cell cycle) and <t>BrdU</t> (a marker labeling cells entering into S phase) revealed a significant decrease in the number of proliferating (Ki-67 + or BrdU + ) cells per unit area in the ribcage and ventral midline of the null FlnA and null FlnA+Fmn2 embryos compared to control WT embryo. Brackets mark the thickness of the ribcage, whereas the rectangular box delineates the thickness of the ventral midline. B. The number of BrdU or Ki67 positive cells in the ventral midline of null FlnA and null FlnA+Fmn2 embryos was decreased by more than 50% in comparison with those in WT embryo (WT vs null FlnA *** = p
    Mouse Anti Flna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Illumina Inc flna editing levels
    Loss of <t>FlnA</t> and Fmn2 leads to a defect in cell proliferation along the ventral thoracic wall. A. Fluorescent photomicrographs of immunostaining performed on transverse sections of E13.5 mouse thorax for proliferation markers. Ki-67 (a marker for cells in the cell cycle) and <t>BrdU</t> (a marker labeling cells entering into S phase) revealed a significant decrease in the number of proliferating (Ki-67 + or BrdU + ) cells per unit area in the ribcage and ventral midline of the null FlnA and null FlnA+Fmn2 embryos compared to control WT embryo. Brackets mark the thickness of the ribcage, whereas the rectangular box delineates the thickness of the ventral midline. B. The number of BrdU or Ki67 positive cells in the ventral midline of null FlnA and null FlnA+Fmn2 embryos was decreased by more than 50% in comparison with those in WT embryo (WT vs null FlnA *** = p
    Flna Editing Levels, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Ambry Genetics flna
    Loss of <t>FlnA</t> and Fmn2 leads to a defect in cell proliferation along the ventral thoracic wall. A. Fluorescent photomicrographs of immunostaining performed on transverse sections of E13.5 mouse thorax for proliferation markers. Ki-67 (a marker for cells in the cell cycle) and <t>BrdU</t> (a marker labeling cells entering into S phase) revealed a significant decrease in the number of proliferating (Ki-67 + or BrdU + ) cells per unit area in the ribcage and ventral midline of the null FlnA and null FlnA+Fmn2 embryos compared to control WT embryo. Brackets mark the thickness of the ribcage, whereas the rectangular box delineates the thickness of the ventral midline. B. The number of BrdU or Ki67 positive cells in the ventral midline of null FlnA and null FlnA+Fmn2 embryos was decreased by more than 50% in comparison with those in WT embryo (WT vs null FlnA *** = p
    Flna, supplied by Ambry Genetics, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Functional analyses of the exon 40-skipped FLNA. ( a ) GST pull-down assay of FLNA with GST-fused Integrin β7 (GST-ITG). WT, wild-type FLNA; 4del, truncated FLNA and skip, FLNA internally lacking 41 AAs. Representative results of three independent

    Journal: European Journal of Human Genetics

    Article Title: Exon skipping causes atypical phenotypes associated with a loss-of-function mutation in FLNA by restoring its protein function

    doi: 10.1038/ejhg.2015.119

    Figure Lengend Snippet: Functional analyses of the exon 40-skipped FLNA. ( a ) GST pull-down assay of FLNA with GST-fused Integrin β7 (GST-ITG). WT, wild-type FLNA; 4del, truncated FLNA and skip, FLNA internally lacking 41 AAs. Representative results of three independent

    Article Snippet: Proteins were analyzed by SDS-PAGE, followed by western blotting using anti-FLNA (MAB1680, Millipore) and anti-GST (sc-138, Santa Cruz Biotechnology) antibodies.

    Techniques: Functional Assay, Pull Down Assay, Atomic Absorption Spectroscopy

    Expression of endogenous FLNa fragments and localization of C-terminal fragment of FLNa. ( A ) Immunoblot of FLNa fragments from HeLa, FLNa-repleted A7, FLNa-deficient M2, prostate carcinoma PC3, and DU145, primary genital skin fibroblasts containing normal AR [LDC, cultured with (+) or without DHT] or nonfunctional mutant AR (TBL) cell cultures. Cell extracts were probed with an antibody that recognizes FLNa repeats 16–20. Arrows indicate full-length FLNa (280 kDa) and a fragment (100 kDa) corresponding to FLNa(16–24). Immunoreactive bands (≈185 and 160 kDa, marked by *) were observed only in extracts from genital fibroblasts. The cytoskeletal paxillin was used to indicate the presence of proteins in each lane. ( B ) Subcellular localization of GFP-FLNa constructs. Chimeric full-length filamin (FL) or FLNa(16–24, aa1788–2647) linked to GFP was coexpressed with WT AR in the absence (+vehicle) or presence of DHT. Representative green fluorescence images for GFP-FLNa constructs (GFP), red immunofluorescence images for AR (AR), and merged images after superimposition of GFP and AR (merge) are shown. Note that yellow in the merged images indicate colocalization of AR and FLNa(16–24) to the nucleus.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Filamin-A fragment localizes to the nucleus to regulate androgen receptor and coactivator functions

    doi: 10.1073/pnas.0736237100

    Figure Lengend Snippet: Expression of endogenous FLNa fragments and localization of C-terminal fragment of FLNa. ( A ) Immunoblot of FLNa fragments from HeLa, FLNa-repleted A7, FLNa-deficient M2, prostate carcinoma PC3, and DU145, primary genital skin fibroblasts containing normal AR [LDC, cultured with (+) or without DHT] or nonfunctional mutant AR (TBL) cell cultures. Cell extracts were probed with an antibody that recognizes FLNa repeats 16–20. Arrows indicate full-length FLNa (280 kDa) and a fragment (100 kDa) corresponding to FLNa(16–24). Immunoreactive bands (≈185 and 160 kDa, marked by *) were observed only in extracts from genital fibroblasts. The cytoskeletal paxillin was used to indicate the presence of proteins in each lane. ( B ) Subcellular localization of GFP-FLNa constructs. Chimeric full-length filamin (FL) or FLNa(16–24, aa1788–2647) linked to GFP was coexpressed with WT AR in the absence (+vehicle) or presence of DHT. Representative green fluorescence images for GFP-FLNa constructs (GFP), red immunofluorescence images for AR (AR), and merged images after superimposition of GFP and AR (merge) are shown. Note that yellow in the merged images indicate colocalization of AR and FLNa(16–24) to the nucleus.

    Article Snippet: Cells were treated according to experimental designs, and immunoblotting of total cell lysates was performed according to standard protocol by using anti-AR 441 antibody (Santa Cruz Biotechnology), anti-FLNa antibody (Chemicon MAB1680), and anti-paxillin antibody (Chemicon MAB3060).

    Techniques: Expressing, Cell Culture, Mutagenesis, Construct, Fluorescence, Immunofluorescence

    Cell Type-specific Alternative Splicing of Filamin A in Cerebral Cortical Development and Human PVNH A) RNA-Seq reads around Flna exonN showing its higher inclusion in adult cerebral cortex, cerebellum and E14.5 CP, in comparison to E14.5 VZ and other non-neural adult tissues. B) Flna exonN has an in-frame stop codon. C) ExonN inclusion is predicted to truncate Flna protein. D) Blocking of NMD by cycloheximide (CHX) in primary hippocampal neurons cultured in vitro for 1 day increases inclusion of exonN. Data are represented as mean +/− SEM. E) RNA-Seq reads showing the inclusion of exonN and retention of its upstream intron in fetal human CP. F) Filamin A exonN is conserved across representative placental mammals. G) T1 brain MRI shows PVNH (white arrows) in affected individuals PH-01 (male) and PH-04 (female), compared to a healthy control and an unrelated individual with a FLNA null mutation. H) Pedigree showing inheritance of PVNH and the rare c.1429 +182 G > A mutation. I) Sanger sequencing traces showing the rare FLNA variant c.1429+182 G > A. J) A cartoon illustrating the FLNA mini-gene construct and primers used for RT-PCR. N-terminal HA and Flag tags were fused in-frame with exon 8. K) RT-PCR using primer F and R1 (inside exonN) detecting the abnormal exonN inclusion in blood samples of affected individuals. L) RT-PCR (primer pair F-R2) results on FLNA mini-genes expressed in Neuro2a cells, showing that the c.1429+182 G > A mutation caused > 40% of FLNA transcripts to include exonN. M) Western blot of transfected Neuro2a cells detecting the WT mini-FLNA (green, arrow) and the truncated form (arrow head). Red, anti-Gapdh. N) RT-PCR (primer pair F-R2) results of E14.5 mouse brains electroporated FLNA mini-genes on E13.5, showing that the G > A mutation caused abnormal inclusion of exonN. .

    Journal: Cell

    Article Title: Cell Type-specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex

    doi: 10.1016/j.cell.2016.07.025

    Figure Lengend Snippet: Cell Type-specific Alternative Splicing of Filamin A in Cerebral Cortical Development and Human PVNH A) RNA-Seq reads around Flna exonN showing its higher inclusion in adult cerebral cortex, cerebellum and E14.5 CP, in comparison to E14.5 VZ and other non-neural adult tissues. B) Flna exonN has an in-frame stop codon. C) ExonN inclusion is predicted to truncate Flna protein. D) Blocking of NMD by cycloheximide (CHX) in primary hippocampal neurons cultured in vitro for 1 day increases inclusion of exonN. Data are represented as mean +/− SEM. E) RNA-Seq reads showing the inclusion of exonN and retention of its upstream intron in fetal human CP. F) Filamin A exonN is conserved across representative placental mammals. G) T1 brain MRI shows PVNH (white arrows) in affected individuals PH-01 (male) and PH-04 (female), compared to a healthy control and an unrelated individual with a FLNA null mutation. H) Pedigree showing inheritance of PVNH and the rare c.1429 +182 G > A mutation. I) Sanger sequencing traces showing the rare FLNA variant c.1429+182 G > A. J) A cartoon illustrating the FLNA mini-gene construct and primers used for RT-PCR. N-terminal HA and Flag tags were fused in-frame with exon 8. K) RT-PCR using primer F and R1 (inside exonN) detecting the abnormal exonN inclusion in blood samples of affected individuals. L) RT-PCR (primer pair F-R2) results on FLNA mini-genes expressed in Neuro2a cells, showing that the c.1429+182 G > A mutation caused > 40% of FLNA transcripts to include exonN. M) Western blot of transfected Neuro2a cells detecting the WT mini-FLNA (green, arrow) and the truncated form (arrow head). Red, anti-Gapdh. N) RT-PCR (primer pair F-R2) results of E14.5 mouse brains electroporated FLNA mini-genes on E13.5, showing that the G > A mutation caused abnormal inclusion of exonN. .

    Article Snippet: Analysis of Ptbp1 iCLIP-Seq datasets ( ; ) revealed that Ptbp1 bound to the alternatively spliced Flna exonN in both NPCs and C2C12 cells ( ).

    Techniques: RNA Sequencing Assay, Blocking Assay, Cell Culture, In Vitro, Magnetic Resonance Imaging, Mutagenesis, Sequencing, Variant Assay, Construct, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection

    Ptbp1 and Rbfox Proteins Antagonistically Control Neural Progenitor Cell Differentiation A) Immunostaining of E18.5 Ptbp1 conditional knockout (cKO, Nestin-Cre ) cerebral cortex showing typical PVNH (white arrow) and thinner ventricular layer of Pax6+ cells (brackets). B) Western blot of Ptbp1 cKO and control MEF cells showing that proteins level of Ptbp1, Flna and Flnb are decreased in Ptbp1 cKO, while Ptbp2 level is increased. C) RT-PCR results showing that Ptbp1 cKO in MEF cells de-represses the inclusion of Flna exonN and a 98-nt Flnb exon. D) –E) Representative images and statistical analysis E) showing that Ptbp1 knockdown (green) at E13.5 results in reduced neural progenitors in the VZ at E15.5. The defect was partially rescued by co-expression of Ptbp1 coding sequence (CDS, red) or FLNA CDS. Numbers in parentheses indicate the number of embryos analyzed. Data are represented as mean +/− SD. F) –G) Representative images and statistical analysis G) showing that introducing Rbfox3 expression into E13.5 mouse brains resulted in reduced progenitor cells in the VZ and reduced neurons in the CP at E16.5. Ectopic expression of Rbfox3 on top of Ptbp1 knockdown causes a more severe depletion of NPCs in the VZ. Data are represented as mean +/− SD. H) A working model showing that Rbfox1/2/3 proteins are highly expressed in neurons and promote neuronal exon inclusion (red), while Ptbp1 is expressed in NPCs (blue, VZ) and represses neuronal exon inclusion. Sox2 binds to the promoter region of Ptbp1 (left). Dysregulation of Rbfox1/2/3 mediated AS may lead to brain disorders such as autism and intellectual disability. Mutations that de-represses neuronal exon inclusion in NPCs may result in PVNH (through FLNA ). .

    Journal: Cell

    Article Title: Cell Type-specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex

    doi: 10.1016/j.cell.2016.07.025

    Figure Lengend Snippet: Ptbp1 and Rbfox Proteins Antagonistically Control Neural Progenitor Cell Differentiation A) Immunostaining of E18.5 Ptbp1 conditional knockout (cKO, Nestin-Cre ) cerebral cortex showing typical PVNH (white arrow) and thinner ventricular layer of Pax6+ cells (brackets). B) Western blot of Ptbp1 cKO and control MEF cells showing that proteins level of Ptbp1, Flna and Flnb are decreased in Ptbp1 cKO, while Ptbp2 level is increased. C) RT-PCR results showing that Ptbp1 cKO in MEF cells de-represses the inclusion of Flna exonN and a 98-nt Flnb exon. D) –E) Representative images and statistical analysis E) showing that Ptbp1 knockdown (green) at E13.5 results in reduced neural progenitors in the VZ at E15.5. The defect was partially rescued by co-expression of Ptbp1 coding sequence (CDS, red) or FLNA CDS. Numbers in parentheses indicate the number of embryos analyzed. Data are represented as mean +/− SD. F) –G) Representative images and statistical analysis G) showing that introducing Rbfox3 expression into E13.5 mouse brains resulted in reduced progenitor cells in the VZ and reduced neurons in the CP at E16.5. Ectopic expression of Rbfox3 on top of Ptbp1 knockdown causes a more severe depletion of NPCs in the VZ. Data are represented as mean +/− SD. H) A working model showing that Rbfox1/2/3 proteins are highly expressed in neurons and promote neuronal exon inclusion (red), while Ptbp1 is expressed in NPCs (blue, VZ) and represses neuronal exon inclusion. Sox2 binds to the promoter region of Ptbp1 (left). Dysregulation of Rbfox1/2/3 mediated AS may lead to brain disorders such as autism and intellectual disability. Mutations that de-represses neuronal exon inclusion in NPCs may result in PVNH (through FLNA ). .

    Article Snippet: Analysis of Ptbp1 iCLIP-Seq datasets ( ; ) revealed that Ptbp1 bound to the alternatively spliced Flna exonN in both NPCs and C2C12 cells ( ).

    Techniques: Cell Differentiation, Immunostaining, Knock-Out, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Sequencing

    Alternative Exons Showing Higher Inclusion in Neurons Are Antagonistically Regulated by Ptbp1 and Rbfox1/2/3 Proteins A) Unbiased motif analysis of alternative exons with higher inclusion in differentiating neurons reveals the enrichment of CU(C/U)UCUU in the 200 nt 5’ upstream region and GCAU(G/A) in the 200 nt downtream intronic region. B) RNA-Seq results of sorted E14.5 mouse cortical cells showing that Ptbp1 is enriched in NPCs (VZ) and Rbfox1 /2/3 transcripts are enriched in non-VZ cells. Data are represented as mean +/− SEM. C) A scatter plot showing that Ptbp1 knockdown in Neuro2a cells de-represses the inclusion of 24 neuronal exons predicted by motif search in A). D) RT-PCR analysis showing that Ptbp1 knockdown in Neuro2a cells by three different shRNAs promotes inclusion of Flna exonN. E) Western blot and quantified signals showing that Ptbp1 knockdown decreases Flna protein level. Data are represented as mean +/− SEM. )) and RNA-Seq results showing that Ptbp1 binds directly to Flna exonN and its flanking introns in NPCs and C2C12 cells. Red asterisk and dotted line indicate chrX:71,486,253, the homologous nucleotide of human ChrX: 153,594,210 C > T mutation. The arrow indicates direction of transcription. G) Rbfox1/2/3 expression in Neuro2a cells promotes inclusion of 30 neuronal exons identified by motif analyses in A). H) RT-PCR results showing that ectopic expression of Rbfox1 /2/3 promotes inclusion of Nin exon 29. I) Overexpression of Rbfox3 in U2OS cells decreases the protein level of centrosomal Nin (green). Data are represented as median +/− SD. )) showing that Rbfox1/2/3 proteins bind to conserved GCAUG motifs downstream of Nin exon 29 in mouse brains. Vertical red bars on top indicate GCATG sequences. .

    Journal: Cell

    Article Title: Cell Type-specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex

    doi: 10.1016/j.cell.2016.07.025

    Figure Lengend Snippet: Alternative Exons Showing Higher Inclusion in Neurons Are Antagonistically Regulated by Ptbp1 and Rbfox1/2/3 Proteins A) Unbiased motif analysis of alternative exons with higher inclusion in differentiating neurons reveals the enrichment of CU(C/U)UCUU in the 200 nt 5’ upstream region and GCAU(G/A) in the 200 nt downtream intronic region. B) RNA-Seq results of sorted E14.5 mouse cortical cells showing that Ptbp1 is enriched in NPCs (VZ) and Rbfox1 /2/3 transcripts are enriched in non-VZ cells. Data are represented as mean +/− SEM. C) A scatter plot showing that Ptbp1 knockdown in Neuro2a cells de-represses the inclusion of 24 neuronal exons predicted by motif search in A). D) RT-PCR analysis showing that Ptbp1 knockdown in Neuro2a cells by three different shRNAs promotes inclusion of Flna exonN. E) Western blot and quantified signals showing that Ptbp1 knockdown decreases Flna protein level. Data are represented as mean +/− SEM. )) and RNA-Seq results showing that Ptbp1 binds directly to Flna exonN and its flanking introns in NPCs and C2C12 cells. Red asterisk and dotted line indicate chrX:71,486,253, the homologous nucleotide of human ChrX: 153,594,210 C > T mutation. The arrow indicates direction of transcription. G) Rbfox1/2/3 expression in Neuro2a cells promotes inclusion of 30 neuronal exons identified by motif analyses in A). H) RT-PCR results showing that ectopic expression of Rbfox1 /2/3 promotes inclusion of Nin exon 29. I) Overexpression of Rbfox3 in U2OS cells decreases the protein level of centrosomal Nin (green). Data are represented as median +/− SD. )) showing that Rbfox1/2/3 proteins bind to conserved GCAUG motifs downstream of Nin exon 29 in mouse brains. Vertical red bars on top indicate GCATG sequences. .

    Article Snippet: Analysis of Ptbp1 iCLIP-Seq datasets ( ; ) revealed that Ptbp1 bound to the alternatively spliced Flna exonN in both NPCs and C2C12 cells ( ).

    Techniques: RNA Sequencing Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Mutagenesis, Expressing, Over Expression

    Validation of the proteomics results by qPCR and Western blotting. (A) Results of qPCR for the mRNA levels of SDHA, SDHB, RhoGDIα, and FLNA. (B) Results of Western blotting for SDHA, SDHB, RhoGDIα, and FLNA. (C) Densitometric analysis of relative protein expressions, GAPDH was used as internal control. n = 4 per group. ∗ P

    Journal: Frontiers in Physiology

    Article Title: Proteomics Analysis of Myocardial Tissues in a Mouse Model of Coronary Microembolization

    doi: 10.3389/fphys.2018.01318

    Figure Lengend Snippet: Validation of the proteomics results by qPCR and Western blotting. (A) Results of qPCR for the mRNA levels of SDHA, SDHB, RhoGDIα, and FLNA. (B) Results of Western blotting for SDHA, SDHB, RhoGDIα, and FLNA. (C) Densitometric analysis of relative protein expressions, GAPDH was used as internal control. n = 4 per group. ∗ P

    Article Snippet: The membranes were blocked in Tris-buffered saline +0.1% Tween-20 (TBST) containing 5% bovine serum albumin (BSA) for 1 h and then incubated with primary antibody against SDHA, SDHB, RhoGDIα, FLNA (Abcam, United Kingdom) and GAPDH (Cell Signaling Technology, United States) at 4°C overnight.

    Techniques: Real-time Polymerase Chain Reaction, Western Blot

    Cyclin D1 and FLNa co-localize in cell ruffles. MDA-MB-231 cells were stained for cyclin D1 (Green), FLNa (Red) and DNA (blue). Co-localization of cyclin D1 and FLNa is indicated by the yellow color in the merged picture. The scale bar is 10 µm.

    Journal: Cancer research

    Article Title: Cyclin D1/cyclin dependent kinase 4 interacts with filamin A and affects the migration and invasion potential of breast cancer cells

    doi: 10.1158/0008-5472.CAN-08-1108

    Figure Lengend Snippet: Cyclin D1 and FLNa co-localize in cell ruffles. MDA-MB-231 cells were stained for cyclin D1 (Green), FLNa (Red) and DNA (blue). Co-localization of cyclin D1 and FLNa is indicated by the yellow color in the merged picture. The scale bar is 10 µm.

    Article Snippet: Cells were prepared as in the wound healing assay and probed with mouse anti-cyclin D1(DCS-6, 1:50, Cell Signaling Technology, Danvers, MA) and anti-FLNa (1:100, Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies in 1% bovine serum albumin (BSA) at 4°C overnight.

    Techniques: Multiple Displacement Amplification, Staining

    MDA-MB-231 cells were transfected with scrambled, CCND1(51), and/or CCND1(52) siRNA. The amount of cyclin D1 protein was lower after transfection with the cyclin D1-specific siRNA compared to scrambled siRNA. The amount of CDK4 and FLNa was not affected

    Journal: Cancer research

    Article Title: Cyclin D1/cyclin dependent kinase 4 interacts with filamin A and affects the migration and invasion potential of breast cancer cells

    doi: 10.1158/0008-5472.CAN-08-1108

    Figure Lengend Snippet: MDA-MB-231 cells were transfected with scrambled, CCND1(51), and/or CCND1(52) siRNA. The amount of cyclin D1 protein was lower after transfection with the cyclin D1-specific siRNA compared to scrambled siRNA. The amount of CDK4 and FLNa was not affected

    Article Snippet: Cells were prepared as in the wound healing assay and probed with mouse anti-cyclin D1(DCS-6, 1:50, Cell Signaling Technology, Danvers, MA) and anti-FLNa (1:100, Santa Cruz Biotechnology, Santa Cruz, CA) primary antibodies in 1% bovine serum albumin (BSA) at 4°C overnight.

    Techniques: Multiple Displacement Amplification, Transfection

    Increased phosphorylation of FLNA at ser2152 impairs cell migration. ( A ) Brightfield photomicrographs showed that the cells transfected with either Myc or Myc-FLNA migrated to the lower surface of the transwell membrane. The cells were stained with 0.5%

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Brefeldin A-inhibited guanine exchange factor 2 regulates Filamin A phosphorylation and neuronal migration

    doi: 10.1523/JNEUROSCI.1063-12.2012

    Figure Lengend Snippet: Increased phosphorylation of FLNA at ser2152 impairs cell migration. ( A ) Brightfield photomicrographs showed that the cells transfected with either Myc or Myc-FLNA migrated to the lower surface of the transwell membrane. The cells were stained with 0.5%

    Article Snippet: For immunocytochemistry, cells were blocked with 3% goat serum containing 0.25% triton X-100 and stained for primary antibodies against Myc (Sigma), phosphoFLNA ser2152 (gift from F. Nakamura), paxillin (BD Transduction Laboratories) and FLNA (Santa Cruz).

    Techniques: Migration, Transfection, Staining

    Loss of BIG2 enhances FlnA expression and phosphorylation at ser2152. ( A ) Western blot displayed levels of phosphorylated FlnA ser2152 detected in liver, heart, brain, and skin tissues obtained from P0 WT and Arfgef2 −/− mice while total

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Brefeldin A-inhibited guanine exchange factor 2 regulates Filamin A phosphorylation and neuronal migration

    doi: 10.1523/JNEUROSCI.1063-12.2012

    Figure Lengend Snippet: Loss of BIG2 enhances FlnA expression and phosphorylation at ser2152. ( A ) Western blot displayed levels of phosphorylated FlnA ser2152 detected in liver, heart, brain, and skin tissues obtained from P0 WT and Arfgef2 −/− mice while total

    Article Snippet: For immunocytochemistry, cells were blocked with 3% goat serum containing 0.25% triton X-100 and stained for primary antibodies against Myc (Sigma), phosphoFLNA ser2152 (gift from F. Nakamura), paxillin (BD Transduction Laboratories) and FLNA (Santa Cruz).

    Techniques: Expressing, Western Blot, Mouse Assay

    Phosphorylation of FLNA at ser2152 alters FLNA subcellular distribution, its binding affinity to actin fibers and focal adhesion structure. ( A ) CHP100 cells transfected with Myc-FLNA-S2152A or FLNA-S2152D were stained with Myc antibody and phalloidin.

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    Article Title: Brefeldin A-inhibited guanine exchange factor 2 regulates Filamin A phosphorylation and neuronal migration

    doi: 10.1523/JNEUROSCI.1063-12.2012

    Figure Lengend Snippet: Phosphorylation of FLNA at ser2152 alters FLNA subcellular distribution, its binding affinity to actin fibers and focal adhesion structure. ( A ) CHP100 cells transfected with Myc-FLNA-S2152A or FLNA-S2152D were stained with Myc antibody and phalloidin.

    Article Snippet: For immunocytochemistry, cells were blocked with 3% goat serum containing 0.25% triton X-100 and stained for primary antibodies against Myc (Sigma), phosphoFLNA ser2152 (gift from F. Nakamura), paxillin (BD Transduction Laboratories) and FLNA (Santa Cruz).

    Techniques: Binding Assay, Transfection, Staining

    Co-purification of FLNA and integrin β1 and the effects of down-regulation of PrP on integrin β1 and FLNA association. A , left panels , immunoblots ( IM ) show that PrP co-purifies ( IP ) with FLNA, and integrin β1 co-purifies with

    Journal: The Journal of Biological Chemistry

    Article Title: Pro-prion Binds Filamin A, Facilitating Its Interaction with Integrin ?1, and Contributes to Melanomagenesis *

    doi: 10.1074/jbc.M110.147413

    Figure Lengend Snippet: Co-purification of FLNA and integrin β1 and the effects of down-regulation of PrP on integrin β1 and FLNA association. A , left panels , immunoblots ( IM ) show that PrP co-purifies ( IP ) with FLNA, and integrin β1 co-purifies with

    Article Snippet: Mouse anti-FLNA, anti-human integrin β1, anti-talin, and anti-actin mAbs were purchased from Chemicon.

    Techniques: Copurification, Western Blot

    shRNA of FLNC specifically inhibits FLNC expression in shRNA infected ESCC cell lines ( A ) Infection efficiency of shRNA lentiviral vectors in the ESCC cell lines (TE-1 and TE-8). Infection efficiency of shRNA lentiviral vectors was confirmed by visualization of green fluorescent protein (GFP) with a confocal microscope. Nuclei are stained with DAPI to visualize the cells. More than 90% of cells were confirmed to be infected with shRNA lentivirus. ( B , C ) mRNA and protein expression levels of FLNA, FLNB, and FLNC were examined by real-time PCR and immunoblotting. GAPDH was used as a loading control. The FLNA and FLNB expression were not reduced in FLNC shRNA infected cells, and were nearly identical to those of SshRNA infected cells. FLNC expression was clearly inhibited in FLNC shRNA infected cells as compared with its expression in SshRNA infected cells. Densities of the immunoblot bands were quantified using Image J software and normalized to GAPDH to obtain the relative densities (RD).

    Journal: Oncotarget

    Article Title: Filamin C promotes lymphatic invasion and lymphatic metastasis and increases cell motility by regulating Rho GTPase in esophageal squamous cell carcinoma

    doi: 10.18632/oncotarget.14087

    Figure Lengend Snippet: shRNA of FLNC specifically inhibits FLNC expression in shRNA infected ESCC cell lines ( A ) Infection efficiency of shRNA lentiviral vectors in the ESCC cell lines (TE-1 and TE-8). Infection efficiency of shRNA lentiviral vectors was confirmed by visualization of green fluorescent protein (GFP) with a confocal microscope. Nuclei are stained with DAPI to visualize the cells. More than 90% of cells were confirmed to be infected with shRNA lentivirus. ( B , C ) mRNA and protein expression levels of FLNA, FLNB, and FLNC were examined by real-time PCR and immunoblotting. GAPDH was used as a loading control. The FLNA and FLNB expression were not reduced in FLNC shRNA infected cells, and were nearly identical to those of SshRNA infected cells. FLNC expression was clearly inhibited in FLNC shRNA infected cells as compared with its expression in SshRNA infected cells. Densities of the immunoblot bands were quantified using Image J software and normalized to GAPDH to obtain the relative densities (RD).

    Article Snippet: Drugs, reagents, and antibodies The following reagents were purchased from the indicated manufacturers: RPMI 1640 (Nikken Biomedical Laboratory, Osaka, Japan); fetal calf serum (FCS) (PAA Laboratories, Pasching, Austria); MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (Sigma-Aldrich, St. Louis, MO, USA); DAPI (4′,6-Diamidino-2-phenylindole, dihydrochloride, solution) (Dojindo Laboratories, Kumamoto, Japan); monoclonal antibodies against FLNA, GAPDH (EMD Millipore, Billerica, MA, USA; Cell Signaling Technology, Danvers, MA, USA, respectively), polyclonal antibodies against FLNB, FLNC (EMD Millipore, Billerica, MA, USA; Atlas Antibodies, Stockholm, Sweden, respectively).

    Techniques: shRNA, Expressing, Infection, Microscopy, Staining, Real-time Polymerase Chain Reaction, Software

    FlnA physically interacts with RhoA and regulates RhoA activation to direct neural progenitor proliferation and differentiation. ( A ) Rho GTPase pull-down assays shows that only active (ACT) or WT-RhoA but not Cdc42 or Rac1 pulls down FlnA by Western blot (left panel) in Neuro2A cells. Conversely, RhoA is co-immunoprecipitated by FlnA C-terminal whereas WT or active Cdc42 and Rac1 did not show any significant binding interaction on Western blot (middle panel). Lastly, full-length FLNA can be co-immunoprecipitated by Flag-RhoA, whereas Flag-Rac1 and Cdc42 cannot (right panel). SUP = supernatant, LAD = molecular weight ladder. Control reflects empty FLAG vector lacking the Rho GTPase constructs followed by pull-down with anti-FLAG antibody and probing for FlnA. ( B ) Fluorescent photomicrographs of cerebral cortex demonstrate FlnA overlapping expression with RhoA in E14 brain cortex, especially, along the apical lining by co-immunostaining and at focal adhesion sites (arrows) of neuroblastoma cells. ( C ) FlnA loss impairs RhoA activation. At baseline, activated RhoA levels are diminished in null FlnA mice. Following laminin activation, RhoA GTPase dramatically increases in the FlnA y/− neural progenitors by 30 min but also declines more precipitously than WT progenitors at 60 min. WT and FlnA y/− neural progenitors are plated on uncoated (uncoated con) or laminin-coated dishes and RhoA activation is assessed at varied time points (30 and 60 min post plating). Upper band corresponds to activated RhoA, and lower band reflects total RhoA (T-RhoA) for loading control. The graph at the right represents mean ± SD values from 5 independent Western blot assays. ( D ) Fluorescent photomicrographs of E14 cerebral cortices 24 h after in utero electroporation of control GFP or dominant negative RhoA (DN-RhoA). Fewer neural cells expressing the DN-RhoA construct reach the cortical plate from the ventricular zone within this time frame, suggesting either impaired migration or diminished neurogenesis. The electroporations specifically target mitotic, proliferating progenitors at the ventricular lining. Progenitors which are therefore GFP + but Ki67- have exited the cell cycle. Higher magnification images and statistical analyses are seen to the right. Co-immunostaining was performed with anti-Ki-67 (marker of cells that remain in the cell cycle) and GFP (marker that initially captures proliferating progenitors) antibodies. The number of GFP + cells that are situated above the ventricular zone is increased in the electroporated GFP control mice (brackets in higher magnification), consistent with increased differentiation. ( E ) The GFP + ,Ki-67 + cells and total GFP + cells from same position-matched area of cortices were quantified. The statistical analysis ( n > 4) show that the ratio of the GFP + , Ki-67 + cells in total GFP + cells is increased approximately 25% in the DN-RhoA electroporation construct versus control. These observations mirror the FlnA loss of function cortical phenotypes and reinforce the likely functional interaction between FlnA and RhoA. Scale bar = 50 μm. ( F ) Fluorescent photomicrographs of GFP (fluorescein) expressing neural cells following electroporation of control GFP or DN-RhoA into lateral ventricles of E13.5 cerebral cortex and staining for early neuronal markers (Tuj1). Acute RhoA inhibition leads to reduction of early born neurons within 72 h, similar to that seen with loss of FlnA. ( G ) Quantification of neurogenic fates is graphically summarized. * = P

    Journal: Cerebral Cortex (New York, NY)

    Article Title: Cytoskeletal Associated Filamin A and RhoA Affect Neural Progenitor Specification During Mitosis

    doi: 10.1093/cercor/bhy033

    Figure Lengend Snippet: FlnA physically interacts with RhoA and regulates RhoA activation to direct neural progenitor proliferation and differentiation. ( A ) Rho GTPase pull-down assays shows that only active (ACT) or WT-RhoA but not Cdc42 or Rac1 pulls down FlnA by Western blot (left panel) in Neuro2A cells. Conversely, RhoA is co-immunoprecipitated by FlnA C-terminal whereas WT or active Cdc42 and Rac1 did not show any significant binding interaction on Western blot (middle panel). Lastly, full-length FLNA can be co-immunoprecipitated by Flag-RhoA, whereas Flag-Rac1 and Cdc42 cannot (right panel). SUP = supernatant, LAD = molecular weight ladder. Control reflects empty FLAG vector lacking the Rho GTPase constructs followed by pull-down with anti-FLAG antibody and probing for FlnA. ( B ) Fluorescent photomicrographs of cerebral cortex demonstrate FlnA overlapping expression with RhoA in E14 brain cortex, especially, along the apical lining by co-immunostaining and at focal adhesion sites (arrows) of neuroblastoma cells. ( C ) FlnA loss impairs RhoA activation. At baseline, activated RhoA levels are diminished in null FlnA mice. Following laminin activation, RhoA GTPase dramatically increases in the FlnA y/− neural progenitors by 30 min but also declines more precipitously than WT progenitors at 60 min. WT and FlnA y/− neural progenitors are plated on uncoated (uncoated con) or laminin-coated dishes and RhoA activation is assessed at varied time points (30 and 60 min post plating). Upper band corresponds to activated RhoA, and lower band reflects total RhoA (T-RhoA) for loading control. The graph at the right represents mean ± SD values from 5 independent Western blot assays. ( D ) Fluorescent photomicrographs of E14 cerebral cortices 24 h after in utero electroporation of control GFP or dominant negative RhoA (DN-RhoA). Fewer neural cells expressing the DN-RhoA construct reach the cortical plate from the ventricular zone within this time frame, suggesting either impaired migration or diminished neurogenesis. The electroporations specifically target mitotic, proliferating progenitors at the ventricular lining. Progenitors which are therefore GFP + but Ki67- have exited the cell cycle. Higher magnification images and statistical analyses are seen to the right. Co-immunostaining was performed with anti-Ki-67 (marker of cells that remain in the cell cycle) and GFP (marker that initially captures proliferating progenitors) antibodies. The number of GFP + cells that are situated above the ventricular zone is increased in the electroporated GFP control mice (brackets in higher magnification), consistent with increased differentiation. ( E ) The GFP + ,Ki-67 + cells and total GFP + cells from same position-matched area of cortices were quantified. The statistical analysis ( n > 4) show that the ratio of the GFP + , Ki-67 + cells in total GFP + cells is increased approximately 25% in the DN-RhoA electroporation construct versus control. These observations mirror the FlnA loss of function cortical phenotypes and reinforce the likely functional interaction between FlnA and RhoA. Scale bar = 50 μm. ( F ) Fluorescent photomicrographs of GFP (fluorescein) expressing neural cells following electroporation of control GFP or DN-RhoA into lateral ventricles of E13.5 cerebral cortex and staining for early neuronal markers (Tuj1). Acute RhoA inhibition leads to reduction of early born neurons within 72 h, similar to that seen with loss of FlnA. ( G ) Quantification of neurogenic fates is graphically summarized. * = P

    Article Snippet: The following antibodies with corresponding dilutions were used for the studies: mouse anti-BrdU (1:100, Calbiochem) and rat anti-BrdU (1:150, Serotec, cat. MCA2060), rabbit anti-Ki-67 monoclonal antibody (1:200, Epitomics, cat. 4203-1), rabbit anti-PH3 polyclonal antibody (1:250, Millipore cat: 06–570), rabbit anti-Sox2 (1:250, Epitomics, cat. 2683-1), rabbit anti-Tbr-1 and anti-Tbr-2 (1:150, Abcam, cat. ab31940, ab23345), mouse anti-E-cadherin and anti-N-cadherin (1:100, BD Biosciences, cat. 610181 and 610920), α-tubulin (Santa Cruz, cat. 32293), rat anti-β1 integrin (1:100, Millipore, cat: MAB 1997), FLNA (1:200, Epitomics cat. 2242-1), mouse ant-RhoA(1:50, Santa Cruz, clone:26C4, cat: sc-418) and rat anti-RhoA (clone: lulu51) was gifted from Dr Shigenobu Yonemura at RIKEN Center for Developmental Biology, mouse anti-Tuj1 (1:200, Covance cat: MMS-435P), mouse anti-aurora b (AIM-1, 1:100, BD Bioscience, cat. 611082), cdk1(1:1000, Calbiochem, pc25), Alexa fluor 488 or rhodamine-labeled phalloidin (1:50, Invitrogen cat. A12379 and R415).

    Techniques: Activation Assay, Western Blot, Immunoprecipitation, Binding Assay, Molecular Weight, Plasmid Preparation, Construct, Expressing, Immunostaining, Mouse Assay, In Utero, Electroporation, Dominant Negative Mutation, Migration, Marker, Functional Assay, Staining, Inhibition

    Cyclin D1 and FLNa co-localize in cell ruffles. MDA-MB-231 cells were stained for cyclin D1 (Green), FLNa (Red) and DNA (blue). Co-localization of cyclin D1 and FLNa is indicated by the yellow color in the merged picture. The scale bar is 10 µm.

    Journal: Cancer research

    Article Title: Cyclin D1/cyclin dependent kinase 4 interacts with filamin A and affects the migration and invasion potential of breast cancer cells

    doi: 10.1158/0008-5472.CAN-08-1108

    Figure Lengend Snippet: Cyclin D1 and FLNa co-localize in cell ruffles. MDA-MB-231 cells were stained for cyclin D1 (Green), FLNa (Red) and DNA (blue). Co-localization of cyclin D1 and FLNa is indicated by the yellow color in the merged picture. The scale bar is 10 µm.

    Article Snippet: The following antibodies were used for Western blotting: anti-cyclin D1 (Ab-3) polyclonal antibody (Lab Vision/Neomarker, Fremont, CA); anti-cyclin D1 (DCS-6) monoclonal antibody, anti-CDK4 (C-22) and anti-actin (C-11) polyclonal antibodies, anti-FLNa and anti-phospho-FLNa (Ser2152) polyclonal antibodies (Cell Signaling Technology, Danvers, MA); and ImmunoPure horseradish peroxidase conjugated goat anti-rabbit antibodies (Pierce Biotechnology, Rockford, IL).

    Techniques: Multiple Displacement Amplification, Staining

    MDA-MB-231 cells were transfected with scrambled, CCND1(51), and/or CCND1(52) siRNA. The amount of cyclin D1 protein was lower after transfection with the cyclin D1-specific siRNA compared to scrambled siRNA. The amount of CDK4 and FLNa was not affected

    Journal: Cancer research

    Article Title: Cyclin D1/cyclin dependent kinase 4 interacts with filamin A and affects the migration and invasion potential of breast cancer cells

    doi: 10.1158/0008-5472.CAN-08-1108

    Figure Lengend Snippet: MDA-MB-231 cells were transfected with scrambled, CCND1(51), and/or CCND1(52) siRNA. The amount of cyclin D1 protein was lower after transfection with the cyclin D1-specific siRNA compared to scrambled siRNA. The amount of CDK4 and FLNa was not affected

    Article Snippet: The following antibodies were used for Western blotting: anti-cyclin D1 (Ab-3) polyclonal antibody (Lab Vision/Neomarker, Fremont, CA); anti-cyclin D1 (DCS-6) monoclonal antibody, anti-CDK4 (C-22) and anti-actin (C-11) polyclonal antibodies, anti-FLNa and anti-phospho-FLNa (Ser2152) polyclonal antibodies (Cell Signaling Technology, Danvers, MA); and ImmunoPure horseradish peroxidase conjugated goat anti-rabbit antibodies (Pierce Biotechnology, Rockford, IL).

    Techniques: Multiple Displacement Amplification, Transfection

    FlnA regulates FlnB expression levels in chondrocytes and vice versa ( A ) Western blot and immunostaining analysis of FlnA and FlnB expression levels in FlnB knockdown and FlnA knockdown ATDC5 cells, respectively. FlnA protein levels are increased with FlnB inhibition. Conversely, FlnB levels are increased with FlnA inhibition. Levels are graphically quantified to the right. ( B ) Fluorescent photomicrograph demonstrates increased FlnA immunostaining (rhodamine) in stably transfected ATDC5 cells, lacking FlnB (FlnBsh), relative to control. Occasional control cells do show increased FlnA expression, with the vast majority exhibiting lower levels of staining. ( C ) Fluorescent photomicrograph demonstrates increased FlnA immunostaining (rhodamine) in stably transfected ATDC5 cells, lacking FlnB (FlnBsh), relative to control. ( D ) Fluorescent photomicrographs of the growth plate from the radius of newborn FlnB -/- mice. Immnostaining shows FlnA (rhodamine) expression is decreased in the hypertrophic zone (double arrows) but the increased in the proliferative zone (single arrow). ( E ) Similarly, FlnB expression (rhodamine) is decreased in the hypertrophic zone but increased in the proliferative zone of FlnA -/- mice. Scale bars in B and C =100 μm, D and E = 200 μm. Quantification is based on n ≥ 3 tissues sections/western blots per experiment and n > 50 cells.

    Journal: Human Molecular Genetics

    Article Title: Opposing FlnA and FlnB interactions regulate RhoA activation in guiding dynamic actin stress fiber formation and cell spreading

    doi: 10.1093/hmg/ddx047

    Figure Lengend Snippet: FlnA regulates FlnB expression levels in chondrocytes and vice versa ( A ) Western blot and immunostaining analysis of FlnA and FlnB expression levels in FlnB knockdown and FlnA knockdown ATDC5 cells, respectively. FlnA protein levels are increased with FlnB inhibition. Conversely, FlnB levels are increased with FlnA inhibition. Levels are graphically quantified to the right. ( B ) Fluorescent photomicrograph demonstrates increased FlnA immunostaining (rhodamine) in stably transfected ATDC5 cells, lacking FlnB (FlnBsh), relative to control. Occasional control cells do show increased FlnA expression, with the vast majority exhibiting lower levels of staining. ( C ) Fluorescent photomicrograph demonstrates increased FlnA immunostaining (rhodamine) in stably transfected ATDC5 cells, lacking FlnB (FlnBsh), relative to control. ( D ) Fluorescent photomicrographs of the growth plate from the radius of newborn FlnB -/- mice. Immnostaining shows FlnA (rhodamine) expression is decreased in the hypertrophic zone (double arrows) but the increased in the proliferative zone (single arrow). ( E ) Similarly, FlnB expression (rhodamine) is decreased in the hypertrophic zone but increased in the proliferative zone of FlnA -/- mice. Scale bars in B and C =100 μm, D and E = 200 μm. Quantification is based on n ≥ 3 tissues sections/western blots per experiment and n > 50 cells.

    Article Snippet: The primary antibodies (for immunostaining and some also for western blotting) were: rabbit anti-FLNA monoclonal antibody (1:300, Cat.# 2242, Epitomics, Burlingame, CA, USA); rabbit anti-FlnB polyclonal antibody (Gifted by Dr. Kao, CWRU).

    Techniques: Expressing, Western Blot, Immunostaining, Inhibition, Stable Transfection, Transfection, Staining, Mouse Assay

    Ndr2-deficiency in murine CD4 + T cells attenuates TCR-induced FLNa phosphorylation at S2152, T-cell adhesion and LFA-1-dependent upregulation of CD69 in vitro . (A) Purified splenic wild type (WT) and Ndr2 −/− CD4 + T cells were left untreated or stimulated with anti-CD3 antibodies for the indicated time points. Lysates were prepared and analyzed by Western blotting using the indicated antibodies. Densitrometric quantification of FLNa phosphorylation at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 3). (B) Purified splenic WT and Ndr2 −/− CD4 + T cells were left untreated (non) or stimulated with anti-CD3 antibodies (CD3) and subsequently analyzed for their ability to bind plate-bound Fc-ICAM-1. Adherent cells were counted and calculated as percentage of input ( n = 3). (C) Purified splenic CD4 + T cells from WT and Ndr2 mice were cultured with plate-bound anti-CD3 antibodies (CD3) in the absence or presence of Fc-ICAM-1 (ICAM-1) with or without blocking LFA-1 antibodies (LFA-1) for 12 h. The upregulation of the activation marker CD69 of unstimulated (0 h) or activated T cells (12 h) were assessed by flow cytometry to determine the mean fluorescence intensity (MFI) ( n = 3). (mean ± SEM; * p ≤ 0.05, ** p ≤ 0.01).

    Journal: Frontiers in Immunology

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells

    doi: 10.3389/fimmu.2018.02852

    Figure Lengend Snippet: Ndr2-deficiency in murine CD4 + T cells attenuates TCR-induced FLNa phosphorylation at S2152, T-cell adhesion and LFA-1-dependent upregulation of CD69 in vitro . (A) Purified splenic wild type (WT) and Ndr2 −/− CD4 + T cells were left untreated or stimulated with anti-CD3 antibodies for the indicated time points. Lysates were prepared and analyzed by Western blotting using the indicated antibodies. Densitrometric quantification of FLNa phosphorylation at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 3). (B) Purified splenic WT and Ndr2 −/− CD4 + T cells were left untreated (non) or stimulated with anti-CD3 antibodies (CD3) and subsequently analyzed for their ability to bind plate-bound Fc-ICAM-1. Adherent cells were counted and calculated as percentage of input ( n = 3). (C) Purified splenic CD4 + T cells from WT and Ndr2 mice were cultured with plate-bound anti-CD3 antibodies (CD3) in the absence or presence of Fc-ICAM-1 (ICAM-1) with or without blocking LFA-1 antibodies (LFA-1) for 12 h. The upregulation of the activation marker CD69 of unstimulated (0 h) or activated T cells (12 h) were assessed by flow cytometry to determine the mean fluorescence intensity (MFI) ( n = 3). (mean ± SEM; * p ≤ 0.05, ** p ≤ 0.01).

    Article Snippet: For Western blotting and immunoprecipitation the following antibodies (Ab) were used: anti-FLAG M2 mAb, anti-β-actin mAb, anti-Talin mAb (clone 8D4) (all from Sigma), anti-Kindlin-3 mAb (Abcam), anti-FLNa mAb, anti-GFP mAb (both from Santa Cruz), anti-FLNa rabbit (Abcam), anti-pFLNa S2152 rabbit Ab, anti-pERK1/2 Thr202/Y204 rabbit Ab, anti-pMst1/2 (Thr183)/(Thr180) rabbit Ab (all from Cell signaling), anti-CD11a mAb (clone 38; Calbiochem Merck), anti-Mst1 mAb (from BD Bioscience) and anti-dsRed mAb (ChromoTek GmbH).

    Techniques: In Vitro, Purification, Western Blot, Mouse Assay, Cell Culture, Blocking Assay, Activation Assay, Marker, Flow Cytometry, Cytometry, Fluorescence

    Activated Ndr2 releases FLNa binding from LFA-1. (A) Jurkat T cells were transfected with constructs that suppress endogenous Ndr2 (shNdr2) and re-express a FLAG-tagged shRNA-resistant wild type (WT Ndr2) or a kinase-dead mutant of Ndr2 (K119A Ndr2). 48 h after transfection, whole-cell extracts were prepared and analyzed by Western blotting using the indicated antibodies. Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of shC-tranfected control cells. (B,C) Cells left untreated or stimulated for the indicated time points with CD3 antibodies. Lysates were used for immunoprecipitation of LFA-1 using anti-CD11a antibodies. Precipitates were divided and analyzed by Western blotting for FLNa, Talin and Kindlin-3 association. Densitrometric analyses of FLNa, Talin, or Kindlin-3 associated to LFA-1 are depicted in Figure S8 .

    Journal: Frontiers in Immunology

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells

    doi: 10.3389/fimmu.2018.02852

    Figure Lengend Snippet: Activated Ndr2 releases FLNa binding from LFA-1. (A) Jurkat T cells were transfected with constructs that suppress endogenous Ndr2 (shNdr2) and re-express a FLAG-tagged shRNA-resistant wild type (WT Ndr2) or a kinase-dead mutant of Ndr2 (K119A Ndr2). 48 h after transfection, whole-cell extracts were prepared and analyzed by Western blotting using the indicated antibodies. Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of shC-tranfected control cells. (B,C) Cells left untreated or stimulated for the indicated time points with CD3 antibodies. Lysates were used for immunoprecipitation of LFA-1 using anti-CD11a antibodies. Precipitates were divided and analyzed by Western blotting for FLNa, Talin and Kindlin-3 association. Densitrometric analyses of FLNa, Talin, or Kindlin-3 associated to LFA-1 are depicted in Figure S8 .

    Article Snippet: For Western blotting and immunoprecipitation the following antibodies (Ab) were used: anti-FLAG M2 mAb, anti-β-actin mAb, anti-Talin mAb (clone 8D4) (all from Sigma), anti-Kindlin-3 mAb (Abcam), anti-FLNa mAb, anti-GFP mAb (both from Santa Cruz), anti-FLNa rabbit (Abcam), anti-pFLNa S2152 rabbit Ab, anti-pERK1/2 Thr202/Y204 rabbit Ab, anti-pMst1/2 (Thr183)/(Thr180) rabbit Ab (all from Cell signaling), anti-CD11a mAb (clone 38; Calbiochem Merck), anti-Mst1 mAb (from BD Bioscience) and anti-dsRed mAb (ChromoTek GmbH).

    Techniques: Binding Assay, Transfection, Construct, shRNA, Mutagenesis, Western Blot, Expressing, Immunoprecipitation

    TCR-stimulation of Jurkat T cells releases LFA-1 from FLNa. (A) Jurkat T cells were left untreated or stimulated for the indicated time points with CD3 antibodies. Lysates were prepared and used for immunoprecipitation of FLNa. Precipitates were analyzed by Western blotting for the phosphorylation status of FLNa at S2152 and LFA-1 association. Aliquots of whole-cell extracts were analyzed for the phosphorylation status of ERK1/2 to verify successful stimulation of T cells (lower panel; Input). (B) Densitrometric analysis of the FLNa phosphorylation status at Serine 2152 (pFLNa) normalized to precipitated total FLNa (tFLNa) or of LFA-1 to precipitated total FLNa (tFLNa) ( n = 4). (mean ± SEM; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

    Journal: Frontiers in Immunology

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells

    doi: 10.3389/fimmu.2018.02852

    Figure Lengend Snippet: TCR-stimulation of Jurkat T cells releases LFA-1 from FLNa. (A) Jurkat T cells were left untreated or stimulated for the indicated time points with CD3 antibodies. Lysates were prepared and used for immunoprecipitation of FLNa. Precipitates were analyzed by Western blotting for the phosphorylation status of FLNa at S2152 and LFA-1 association. Aliquots of whole-cell extracts were analyzed for the phosphorylation status of ERK1/2 to verify successful stimulation of T cells (lower panel; Input). (B) Densitrometric analysis of the FLNa phosphorylation status at Serine 2152 (pFLNa) normalized to precipitated total FLNa (tFLNa) or of LFA-1 to precipitated total FLNa (tFLNa) ( n = 4). (mean ± SEM; * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

    Article Snippet: For Western blotting and immunoprecipitation the following antibodies (Ab) were used: anti-FLAG M2 mAb, anti-β-actin mAb, anti-Talin mAb (clone 8D4) (all from Sigma), anti-Kindlin-3 mAb (Abcam), anti-FLNa mAb, anti-GFP mAb (both from Santa Cruz), anti-FLNa rabbit (Abcam), anti-pFLNa S2152 rabbit Ab, anti-pERK1/2 Thr202/Y204 rabbit Ab, anti-pMst1/2 (Thr183)/(Thr180) rabbit Ab (all from Cell signaling), anti-CD11a mAb (clone 38; Calbiochem Merck), anti-Mst1 mAb (from BD Bioscience) and anti-dsRed mAb (ChromoTek GmbH).

    Techniques: Immunoprecipitation, Western Blot

    Ndr2 phosphorylates FLNa at S2152 in vitro . (A) Purified WT Ndr2/Mob2 heterodimer was used to phosphorylate a positional scanning peptide library using radiolabeled ATP. The degree of phosphorylation of each component of the library, harboring the indicated amino acid residue at the indicated position relative to the phosphorylation site, is shown at left. Quantified data were normalized, log 2 transformed, and used to generate a heat map shown at right ( n = 2). (B) HEK 293T cells were transfected with either empty pEFBOS vector (vector) or plasmids encoding FLAG-tagged wild type Ndr2 (FNdr2) and a kinase dead (K119A) mutant of Ndr2 (FNdr2K119A). Cells were left untreated or treated with okadaic acid (OA), lysed and Ndr2 was immunoprecipitated using FLAG antibodies. A GST-FLNa fragment (19–24 repeats) was used as substrate for an in vitro kinase assay. Reactions were analyzed by Western blotting with the indicated antibodies ( n = 3). (C) Jurkat T cells were left untreated or stimulated for the indicated time points with CD3 antibodies. Cells were lysed and analyzed by Western Blotting with the indicated antibodies. Aliquots of whole-cell extracts were analyzed for the phosphorylation status of ERK1/2 to verify successful stimulation of T cells. Densitrometric analysis of the FLNa phosphorylation status at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 4) (mean ± SEM; ** p ≤ 0.01).

    Journal: Frontiers in Immunology

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells

    doi: 10.3389/fimmu.2018.02852

    Figure Lengend Snippet: Ndr2 phosphorylates FLNa at S2152 in vitro . (A) Purified WT Ndr2/Mob2 heterodimer was used to phosphorylate a positional scanning peptide library using radiolabeled ATP. The degree of phosphorylation of each component of the library, harboring the indicated amino acid residue at the indicated position relative to the phosphorylation site, is shown at left. Quantified data were normalized, log 2 transformed, and used to generate a heat map shown at right ( n = 2). (B) HEK 293T cells were transfected with either empty pEFBOS vector (vector) or plasmids encoding FLAG-tagged wild type Ndr2 (FNdr2) and a kinase dead (K119A) mutant of Ndr2 (FNdr2K119A). Cells were left untreated or treated with okadaic acid (OA), lysed and Ndr2 was immunoprecipitated using FLAG antibodies. A GST-FLNa fragment (19–24 repeats) was used as substrate for an in vitro kinase assay. Reactions were analyzed by Western blotting with the indicated antibodies ( n = 3). (C) Jurkat T cells were left untreated or stimulated for the indicated time points with CD3 antibodies. Cells were lysed and analyzed by Western Blotting with the indicated antibodies. Aliquots of whole-cell extracts were analyzed for the phosphorylation status of ERK1/2 to verify successful stimulation of T cells. Densitrometric analysis of the FLNa phosphorylation status at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 4) (mean ± SEM; ** p ≤ 0.01).

    Article Snippet: For Western blotting and immunoprecipitation the following antibodies (Ab) were used: anti-FLAG M2 mAb, anti-β-actin mAb, anti-Talin mAb (clone 8D4) (all from Sigma), anti-Kindlin-3 mAb (Abcam), anti-FLNa mAb, anti-GFP mAb (both from Santa Cruz), anti-FLNa rabbit (Abcam), anti-pFLNa S2152 rabbit Ab, anti-pERK1/2 Thr202/Y204 rabbit Ab, anti-pMst1/2 (Thr183)/(Thr180) rabbit Ab (all from Cell signaling), anti-CD11a mAb (clone 38; Calbiochem Merck), anti-Mst1 mAb (from BD Bioscience) and anti-dsRed mAb (ChromoTek GmbH).

    Techniques: In Vitro, Purification, Transformation Assay, Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Kinase Assay, Western Blot

    Ndr2 phosphorylates FLNa at S2152 in Jurkat T cells in vivo . (A) Jurkat T cells were transfected with suppression/re-expression constructs which suppress endogenous Ndr2 (shNdr2) and re-express a FLAG-tagged shRNA-resistant wild type (WT Ndr2) or a kinase-dead mutant of Ndr2 (K119A Ndr2). Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of the shC-tranfected control cells. (B) At 48 h post-transfection, cells left untreated or stimulated for the indicated time points with CD3 antibodies. Cells were lysed and analyzed by Western Blotting with the indicated antibodies. Densitrometric quantification of FLNa phosphorylation at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 3). (mean ± SEM; ** p ≤ 0.05).

    Journal: Frontiers in Immunology

    Article Title: Filamin A Phosphorylation at Serine 2152 by the Serine/Threonine Kinase Ndr2 Controls TCR-Induced LFA-1 Activation in T Cells

    doi: 10.3389/fimmu.2018.02852

    Figure Lengend Snippet: Ndr2 phosphorylates FLNa at S2152 in Jurkat T cells in vivo . (A) Jurkat T cells were transfected with suppression/re-expression constructs which suppress endogenous Ndr2 (shNdr2) and re-express a FLAG-tagged shRNA-resistant wild type (WT Ndr2) or a kinase-dead mutant of Ndr2 (K119A Ndr2). Numbers represent the reduction and re-expression of Ndr2 and its mutant after normalization to the Ndr2 expression level of the shC-tranfected control cells. (B) At 48 h post-transfection, cells left untreated or stimulated for the indicated time points with CD3 antibodies. Cells were lysed and analyzed by Western Blotting with the indicated antibodies. Densitrometric quantification of FLNa phosphorylation at Serine 2152 (pFLNa) normalized to total FLNa (tFLNa) ( n = 3). (mean ± SEM; ** p ≤ 0.05).

    Article Snippet: For Western blotting and immunoprecipitation the following antibodies (Ab) were used: anti-FLAG M2 mAb, anti-β-actin mAb, anti-Talin mAb (clone 8D4) (all from Sigma), anti-Kindlin-3 mAb (Abcam), anti-FLNa mAb, anti-GFP mAb (both from Santa Cruz), anti-FLNa rabbit (Abcam), anti-pFLNa S2152 rabbit Ab, anti-pERK1/2 Thr202/Y204 rabbit Ab, anti-pMst1/2 (Thr183)/(Thr180) rabbit Ab (all from Cell signaling), anti-CD11a mAb (clone 38; Calbiochem Merck), anti-Mst1 mAb (from BD Bioscience) and anti-dsRed mAb (ChromoTek GmbH).

    Techniques: In Vivo, Transfection, Expressing, Construct, shRNA, Mutagenesis, Western Blot

    Chemotaxis in response to CCL2 is significantly reduced in THP-1 cells treated with FLNa siRNA. ( A ) Histogram of mean chemotaxis migration results from THP-1 cells treated for 5 days with 100 nM of FLNa siRNA or negative control siRNA. Cells were left to migrate for 24 hours. Experiments were repeated four times. ( B ) Immunoblot of FLNa knock-down in THP-1 cells with 100 nM FLNa siRNA or negative control siRNA as indicated. ( C ) Schematic model of CCL2-activated CCR2B in the cell membrane and during internalization. 1 ) Dimerized FLNa interacts with the C-terminus of the CCR2B through at least repeat 19 and with β-arrestin through repeat 22. β-arrestin also binds the third intracellular loop of CCR2B. In addition, FLNa interacts with actin to crosslink actin fibers . 2 ) Vesicle-budding step of CCR2B-endocytosis. During CCL2-induced clathrin-mediated endocytosis of CCR2B, FLNa links the receptor and arrestin molecules to actin. Actin polymerization is triggered when the clathrin coat and the plasma membrane start to invaginate, and stops when vesicle scission occurs. 3 ) Endogenous vesicles can be propelled through the cytoskeleton by actin comet tails, which might facilitate movement towards cytoskeletal structures or aid the fusion of endocytic organelles.

    Journal: PLoS ONE

    Article Title: Filamin A Binds to CCR2B and Regulates Its Internalization

    doi: 10.1371/journal.pone.0012212

    Figure Lengend Snippet: Chemotaxis in response to CCL2 is significantly reduced in THP-1 cells treated with FLNa siRNA. ( A ) Histogram of mean chemotaxis migration results from THP-1 cells treated for 5 days with 100 nM of FLNa siRNA or negative control siRNA. Cells were left to migrate for 24 hours. Experiments were repeated four times. ( B ) Immunoblot of FLNa knock-down in THP-1 cells with 100 nM FLNa siRNA or negative control siRNA as indicated. ( C ) Schematic model of CCL2-activated CCR2B in the cell membrane and during internalization. 1 ) Dimerized FLNa interacts with the C-terminus of the CCR2B through at least repeat 19 and with β-arrestin through repeat 22. β-arrestin also binds the third intracellular loop of CCR2B. In addition, FLNa interacts with actin to crosslink actin fibers . 2 ) Vesicle-budding step of CCR2B-endocytosis. During CCL2-induced clathrin-mediated endocytosis of CCR2B, FLNa links the receptor and arrestin molecules to actin. Actin polymerization is triggered when the clathrin coat and the plasma membrane start to invaginate, and stops when vesicle scission occurs. 3 ) Endogenous vesicles can be propelled through the cytoskeleton by actin comet tails, which might facilitate movement towards cytoskeletal structures or aid the fusion of endocytic organelles.

    Article Snippet: Some of the samples treated with siRNA were lysed in 0.5% NP-40 lysis buffer and subjected to immunoblotting using rabbit anti-FLNa (Abcam) and anti-β-actin antibodies.

    Techniques: Chemotaxis Assay, Migration, Negative Control

    Interaction between CCR2B and FLNa and subcellular distribution of CCR2B with FLNa in monocytes. ( A ) Co-immunoprecipitation of the Myc-tagged CCR2B tail fragment and full length FLNa . Lysates from A7 and M2 cells were added to the in vitro transcription/translated CCR2B-tail (aa 314–360) and anti-c-Myc was used for immunoprecipitation. The translated parental expression vector was used as control (C). IgG levels are indicated in the lower panel. ( B ) Coimmunoprecipitation of full length CCR2B with endogenous FLNa. HEK293 cells were transfected with pcDNA3-FLAG-CCR2B (2B) or pcDNA3-FLAG (C). M2 anti-FLAG antibodies were used for immunoprecipitation. The CCR2B dimer (D) and monomer (M) are indicated. FLNa and β-actin levels in the lysates are shown in the two lower panels. Blots are representative of at least three independent experiments. ( C ) Mono Mac 1 cells ( upper panel ) and THP-1 ( lower panel ) cells were fixed and incubated with anti-CCR2 and donkey anti-rabbit Alexa Fluor 488 and anti-filamin 1 and Texas Red goat anti-mouse. Images are from one single layer of the Z stacks. The colocalization between CCR2B (green) and FLNa (red) was analyzed using Imaris colocalization software and is shown in white. Experiments were done in duplicates and repeated three times.

    Journal: PLoS ONE

    Article Title: Filamin A Binds to CCR2B and Regulates Its Internalization

    doi: 10.1371/journal.pone.0012212

    Figure Lengend Snippet: Interaction between CCR2B and FLNa and subcellular distribution of CCR2B with FLNa in monocytes. ( A ) Co-immunoprecipitation of the Myc-tagged CCR2B tail fragment and full length FLNa . Lysates from A7 and M2 cells were added to the in vitro transcription/translated CCR2B-tail (aa 314–360) and anti-c-Myc was used for immunoprecipitation. The translated parental expression vector was used as control (C). IgG levels are indicated in the lower panel. ( B ) Coimmunoprecipitation of full length CCR2B with endogenous FLNa. HEK293 cells were transfected with pcDNA3-FLAG-CCR2B (2B) or pcDNA3-FLAG (C). M2 anti-FLAG antibodies were used for immunoprecipitation. The CCR2B dimer (D) and monomer (M) are indicated. FLNa and β-actin levels in the lysates are shown in the two lower panels. Blots are representative of at least three independent experiments. ( C ) Mono Mac 1 cells ( upper panel ) and THP-1 ( lower panel ) cells were fixed and incubated with anti-CCR2 and donkey anti-rabbit Alexa Fluor 488 and anti-filamin 1 and Texas Red goat anti-mouse. Images are from one single layer of the Z stacks. The colocalization between CCR2B (green) and FLNa (red) was analyzed using Imaris colocalization software and is shown in white. Experiments were done in duplicates and repeated three times.

    Article Snippet: Some of the samples treated with siRNA were lysed in 0.5% NP-40 lysis buffer and subjected to immunoblotting using rabbit anti-FLNa (Abcam) and anti-β-actin antibodies.

    Techniques: Immunoprecipitation, In Vitro, Expressing, Plasmid Preparation, Transfection, Incubation, Software

    Model representing the in vivo S6K-mediated phosphorylation of FLNA, which contributes to S6K-dependent cell shape changes. Cell shape changes occur in vivo during both inflammatory processes and cancer progression and the present study supports an active

    Journal: The FASEB Journal

    Article Title: S6K is a morphogenic protein with a mechanism involving Filamin-A phosphorylation and phosphatidic acid binding

    doi: 10.1096/fj.14-260992

    Figure Lengend Snippet: Model representing the in vivo S6K-mediated phosphorylation of FLNA, which contributes to S6K-dependent cell shape changes. Cell shape changes occur in vivo during both inflammatory processes and cancer progression and the present study supports an active

    Article Snippet: Alternatively, to measure S6K activity on FLNA directly, purified, recombinant FLNA (Origene, Rockville, MD, USA) or a synthetic peptide substrate representing the primary FLNA phosphorylation site (5′-RRRAPSVANVG-3′) (Bio-Synthesis, Inc., Lewisville, TX, USA) were used in place of the S6K-RSK2 peptide substate.

    Techniques: In Vivo

    S6K and FLNA form a protein-protein complex that contributes to increased actin polymerization. A ) Schematic cartoon representing the myc tags and both S6K and FLNA protein domains used for co-IP reactions. B ) Co-immunoprecipitation assays of protein-protein

    Journal: The FASEB Journal

    Article Title: S6K is a morphogenic protein with a mechanism involving Filamin-A phosphorylation and phosphatidic acid binding

    doi: 10.1096/fj.14-260992

    Figure Lengend Snippet: S6K and FLNA form a protein-protein complex that contributes to increased actin polymerization. A ) Schematic cartoon representing the myc tags and both S6K and FLNA protein domains used for co-IP reactions. B ) Co-immunoprecipitation assays of protein-protein

    Article Snippet: Alternatively, to measure S6K activity on FLNA directly, purified, recombinant FLNA (Origene, Rockville, MD, USA) or a synthetic peptide substrate representing the primary FLNA phosphorylation site (5′-RRRAPSVANVG-3′) (Bio-Synthesis, Inc., Lewisville, TX, USA) were used in place of the S6K-RSK2 peptide substate.

    Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation

    CCR2B localizes at the tips of actin filaments. CCR2B-A7 ( A ) and CCR2B-M2 ( B ) cells were fixed and incubated with antibodies and analyzed as described before. ( C ) CCR2B-A7 cells were fixed, permeabilized and stained with anti-FLAG Cy3 and FITC-phalloidin. Images are from one single layer of the Z stacks. The colocalization between CCR2B (red) and actin (green) was analyzed using Imaris colocalization software and is shown in white. Bars,10 µm. ( D ) CCR2B-A7 cells were fixed, permeabilized and stained with anti-FLAG Cy3, FITC-phalloidin, mouse anti-FLNa followed by goat anti-mouse Alexa633. Images show pseudo-colors for visualization purposes. Colocalization 1 (β-actin vs. CCR2B) and Colocalization 2 (FLNa vs. CCR2B) were analyzed using Imaris colocalization software and are shown in white. Dotted lines in zoom 1 and zoom 2 mark the direction of the stress fibers. Bars, 10 µm. Experiments were done in duplicates and repeated three times.

    Journal: PLoS ONE

    Article Title: Filamin A Binds to CCR2B and Regulates Its Internalization

    doi: 10.1371/journal.pone.0012212

    Figure Lengend Snippet: CCR2B localizes at the tips of actin filaments. CCR2B-A7 ( A ) and CCR2B-M2 ( B ) cells were fixed and incubated with antibodies and analyzed as described before. ( C ) CCR2B-A7 cells were fixed, permeabilized and stained with anti-FLAG Cy3 and FITC-phalloidin. Images are from one single layer of the Z stacks. The colocalization between CCR2B (red) and actin (green) was analyzed using Imaris colocalization software and is shown in white. Bars,10 µm. ( D ) CCR2B-A7 cells were fixed, permeabilized and stained with anti-FLAG Cy3, FITC-phalloidin, mouse anti-FLNa followed by goat anti-mouse Alexa633. Images show pseudo-colors for visualization purposes. Colocalization 1 (β-actin vs. CCR2B) and Colocalization 2 (FLNa vs. CCR2B) were analyzed using Imaris colocalization software and are shown in white. Dotted lines in zoom 1 and zoom 2 mark the direction of the stress fibers. Bars, 10 µm. Experiments were done in duplicates and repeated three times.

    Article Snippet: After 24 h, cells were incubated with mouse anti-FLAG-M2-Cy3 antibody (Sigma-Aldrich Co.) at 4°C and stimulated with 20 nM CCL2 at 37°C for the times indicated before being fixed, and incubated with goat anti-FLNa (Santa Cruz Biotechnology, Inc.) followed by FITC-conjugated mouse anti-goat (Jackson ImmunoResearch, Suffolk, UK).

    Techniques: Incubation, Staining, Software

    Expression of FLNA rescues the radial migration defect of Rcan1 knockdown neurons. A , Western blot analysis showing that expression of the FLNA-EGFP construct increased FLNA protein in cultured cortical neurons. The 0 DIV cortical neurons were electroporated with pSuper-scramble (NC) or Rcan1 #1 shRNA (#1) in combination with empty vector (Control) or FLNA-EGFP (FLNA). Cells were lysed 4 d (4 DIV) after electroporation for Western blot. β-Actin was used as a loading control. B , Quantification of the data obtained as in A . Data are mean ± SEM; n = 3. C , D , Representative images ( C ) and statistics results ( D ) showing that overexpression of FLNA did not affect neuronal migration at P7. IUE was performed using pCAG-EGFP combined with pCMV-EGFP-N1 empty vector (Control) or pcDNA3-FLNA-EGFP (FLNA). Scale bar, 300 μm. Quantification (mean ± SEM) of EGFP-positive cell distribution in the cortices of P7. n = 4 for control; n = 5 for FLNA; t test. E , F , Representative images ( E ) and statistics results ( F ) showing that FLNA could remarkably rescue the migration defect and neuronal clustering caused by Rcan1 knockdown. Rescue experiment was performed by using Rcan1 #1 shRNA combined with pCMV-EGFP-N1 empty vector (Control) or pcDNA3-FLNA-EGFP (FLNA). Scale bars: E , Top, 300 μm; E , Bottom, 50 μm. Quantification (mean ± SEM) of EGFP-positive cell distribution in the cortices of P7. n = 9 for control; n = 4 for FLNA. * p

    Journal: The Journal of Neuroscience

    Article Title: Rcan1 Deficiency Impairs Neuronal Migration and Causes Periventricular Heterotopia

    doi: 10.1523/JNEUROSCI.1003-14.2015

    Figure Lengend Snippet: Expression of FLNA rescues the radial migration defect of Rcan1 knockdown neurons. A , Western blot analysis showing that expression of the FLNA-EGFP construct increased FLNA protein in cultured cortical neurons. The 0 DIV cortical neurons were electroporated with pSuper-scramble (NC) or Rcan1 #1 shRNA (#1) in combination with empty vector (Control) or FLNA-EGFP (FLNA). Cells were lysed 4 d (4 DIV) after electroporation for Western blot. β-Actin was used as a loading control. B , Quantification of the data obtained as in A . Data are mean ± SEM; n = 3. C , D , Representative images ( C ) and statistics results ( D ) showing that overexpression of FLNA did not affect neuronal migration at P7. IUE was performed using pCAG-EGFP combined with pCMV-EGFP-N1 empty vector (Control) or pcDNA3-FLNA-EGFP (FLNA). Scale bar, 300 μm. Quantification (mean ± SEM) of EGFP-positive cell distribution in the cortices of P7. n = 4 for control; n = 5 for FLNA; t test. E , F , Representative images ( E ) and statistics results ( F ) showing that FLNA could remarkably rescue the migration defect and neuronal clustering caused by Rcan1 knockdown. Rescue experiment was performed by using Rcan1 #1 shRNA combined with pCMV-EGFP-N1 empty vector (Control) or pcDNA3-FLNA-EGFP (FLNA). Scale bars: E , Top, 300 μm; E , Bottom, 50 μm. Quantification (mean ± SEM) of EGFP-positive cell distribution in the cortices of P7. n = 9 for control; n = 4 for FLNA. * p

    Article Snippet: Membranes were blocked with 5% no-fat milk in 0.05% Tween 20 at room temperature for 1 h and probed with rabbit anti-Rcan1 (Sigma), rabbit anti-calcineurin (Santa Cruz Biotechnology), HRP-coupled mouse anti-β-actin (Santa Cruz Biotechnology), and rabbit anti-Flna (Epitomics) antibodies.

    Techniques: Expressing, Migration, Western Blot, Construct, Cell Culture, shRNA, Plasmid Preparation, Electroporation, Over Expression

    A model for the role of Rcan1 in neuronal migration. Rcan1 regulates Flna expression at the mRNA level. Knockdown of Rcan1 leads to decreased Flna mRNA, which in turn decreases Flna protein and impairs neuronal migration. The exact mechanism through which Rcan1 regulates Flna mRNA is still unknown, which may be involved in the transcription or stabilization of Flna mRNA. Although Rcan1 regulates the phosphatase activity of calcineurin, this procedure does not participate in the regulation of Flna mRNA.

    Journal: The Journal of Neuroscience

    Article Title: Rcan1 Deficiency Impairs Neuronal Migration and Causes Periventricular Heterotopia

    doi: 10.1523/JNEUROSCI.1003-14.2015

    Figure Lengend Snippet: A model for the role of Rcan1 in neuronal migration. Rcan1 regulates Flna expression at the mRNA level. Knockdown of Rcan1 leads to decreased Flna mRNA, which in turn decreases Flna protein and impairs neuronal migration. The exact mechanism through which Rcan1 regulates Flna mRNA is still unknown, which may be involved in the transcription or stabilization of Flna mRNA. Although Rcan1 regulates the phosphatase activity of calcineurin, this procedure does not participate in the regulation of Flna mRNA.

    Article Snippet: Membranes were blocked with 5% no-fat milk in 0.05% Tween 20 at room temperature for 1 h and probed with rabbit anti-Rcan1 (Sigma), rabbit anti-calcineurin (Santa Cruz Biotechnology), HRP-coupled mouse anti-β-actin (Santa Cruz Biotechnology), and rabbit anti-Flna (Epitomics) antibodies.

    Techniques: Migration, Expressing, Activity Assay

    Loss of FlnA and Fmn2 leads to a defect in cell proliferation along the ventral thoracic wall. A. Fluorescent photomicrographs of immunostaining performed on transverse sections of E13.5 mouse thorax for proliferation markers. Ki-67 (a marker for cells in the cell cycle) and BrdU (a marker labeling cells entering into S phase) revealed a significant decrease in the number of proliferating (Ki-67 + or BrdU + ) cells per unit area in the ribcage and ventral midline of the null FlnA and null FlnA+Fmn2 embryos compared to control WT embryo. Brackets mark the thickness of the ribcage, whereas the rectangular box delineates the thickness of the ventral midline. B. The number of BrdU or Ki67 positive cells in the ventral midline of null FlnA and null FlnA+Fmn2 embryos was decreased by more than 50% in comparison with those in WT embryo (WT vs null FlnA *** = p

    Journal: PLoS ONE

    Article Title: FilaminA and Formin2 regulate skeletal, muscular, and intestinal formation through mesenchymal progenitor proliferation

    doi: 10.1371/journal.pone.0189285

    Figure Lengend Snippet: Loss of FlnA and Fmn2 leads to a defect in cell proliferation along the ventral thoracic wall. A. Fluorescent photomicrographs of immunostaining performed on transverse sections of E13.5 mouse thorax for proliferation markers. Ki-67 (a marker for cells in the cell cycle) and BrdU (a marker labeling cells entering into S phase) revealed a significant decrease in the number of proliferating (Ki-67 + or BrdU + ) cells per unit area in the ribcage and ventral midline of the null FlnA and null FlnA+Fmn2 embryos compared to control WT embryo. Brackets mark the thickness of the ribcage, whereas the rectangular box delineates the thickness of the ventral midline. B. The number of BrdU or Ki67 positive cells in the ventral midline of null FlnA and null FlnA+Fmn2 embryos was decreased by more than 50% in comparison with those in WT embryo (WT vs null FlnA *** = p

    Article Snippet: The following antibodies with corresponding dilutions were used for tissue and cell staining: mouse anti-BrdU (1:100, Calbiochem), rabbit anti-Ki-67 monoclonal antibody (1:200, Epitomics, cat. 4203–1), mouse anti-FLNA (1:100, Santa Cruz sc-58764), mouse anti-Desmin (1:100, BD Bioscience 550626), and rabbit anti-caspase3 (1:100, Cell Signaling technology 9661).

    Techniques: Immunostaining, Marker, Labeling