Journal: bioRxiv

Article Title: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

doi: 10.1101/2020.05.11.083030

Figure Lengend Snippet: (A) Still images from live cell imaging showing NHSL1-EGFP co-expressed with mScarlet-I-tagged Abi1 and (B) Nap1 in B16-F1 cells. Representative images shown from three independent experiments. Scale bar represent 20 µm. Inset is a magnified view of the white box. Scale bar in inset (A) applies to both insets (A,B): 1.25 μm. See also related video S6. (C-E) Endogenous NHSL1 co-localises with Abi1 (C, NHSL1 pAb) Scar/WAVE1 (D, NHSL1 pAb ) , and Scar/WAVE2 (E, NHSL1 mAb ) at the very edge of lamellipodia in B16-F1 mouse melanoma cells. Scale bar in (C) applies also to (D,E): 20 µm. Inset represents a magnified view of the white box. Scale bar in inset in (C) applies also to inset for (D,E): 20 μm. Representative images shown from three independent experiments. See Fig. S8 for line scans of co-localisations in (C) and (E).

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Abi2 (Epitomics, 3189-1), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), ARPC2 (07-227-I-100UG, Millipore).

Techniques: Live Cell Imaging

Journal: bioRxiv

Article Title: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

doi: 10.1101/2020.05.11.083030

Figure Lengend Snippet: (A) The Scar/WAVE complex co-immunoprecipitates with NHSL1. HEK cells were transfected with EGFP-NHSL1, and Myc-Pir121, -Nap-1, -WAVE2, -Abi2. NHSL-1 was immunoprecipitated (pAb 4457) from lysates and co-immunoprecipitation tested on a western blot with Myc and EGFP antibodies. Representative blots from three independent experiments (see Fig. S9A,B for full western blots). (B) and (C) Western blot showing endogenous NHSL1 pulled down with polyclonal NHSL1 (4457) antibody followed by western blotting with (B) Abi1 and (C) Scar/WAVE2 antibodies detecting endogenous co-immunoprecipitation of these proteins in MCF10A (Abi1) and B16-F1 (Scar/WAVE2) cell lysates. Immunoprecipitation using non-immune rabbit IgG served as a negative control. Representative blots from three independent experiments. (D) GST-pull downs using purified Glutathione-sepharose coupled GST-fusion proteins of Abi1 and c-Abl SH3 domains or GST alone from HEK cell lysates that were transfected with EGFP-NHSL1. Following GST-pulldown EGFP-NHSL1 was detected in a western blot with anti-EGFP antibodies. Ponceau staining of membrane reveals GST or GST-tagged Abi- or Abl-SH3 domains used. Representative blots from three independent experiments. (E) Western blot showing immunoprecipitation using NHSL1 polyclonal (4457) antibody or non-immune rabbit IgG control from HEK cell lysates expressing Myc-NHSL1 and all Myc-tagged components of the Scar/WAVE complex, including Myc-Abi1-full-length (E, left), Myc-Abi1-delta-SH3 (E, right). The western blot shows co-immunoprecipitation between NHSL1 and all components of the Scar/WAVE complex only when the Abi SH3 domain is present (Myc-HSPC300 is not shown). Representative blots from three independent experiments. (F) Quantification of band intensity from chemiluminescence from (E) imaged with a CCD camera. Co-immunoprecipitation is reduced by >80%. Bars indicate mean ± SEM (error bars); n = 3; One-way ANOVA: p=0.0002; F(7,16)=8.855; Sidak’s multiple comparisons test: PIR121 **, p=0.0093; Nap1 **, p=0.0028; WAVE2 **, p=0.0074; Abi1 **, p=0.0030. (G) HEK cells were transfected with EGFP-tagged NHSL1 or NHSL1-SH3 binding mutants or EGFP only as negative control and Myc-Abi1. After GFP-trap pulldown of wild type (WT) NHSL1 or different NHSL1-SH3 binding mutants co-precipitation was detected in a western blot with Myc antibody. Representative blots from five independent experiments. Source data are provided as a Source Data file.

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Abi2 (Epitomics, 3189-1), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), ARPC2 (07-227-I-100UG, Millipore).

Techniques: Transfection, Immunoprecipitation, Western Blot, Negative Control, Purification, Staining, Expressing, Binding Assay

Journal: Oncogenesis

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

doi: 10.1038/oncsis.2012.28

Figure Lengend Snippet: Identification of Abi1 mutations in primary prostate tissue. ( a ) Diagram of ABI1/SSH3BP1 gene with identified prostate tumor mutations. Boxes indicate coding region of consecutive exons (1–12 as indicated) of ABI1/SSH3BP1 gene with exonic mutation listed above boxes. Deletions encompassing entire exons (exon 6 and exon 10) are indicated by lines. Numbers below boxes depict the last amino acid encoded by the specific exon or encoded by a shared codon from two consecutive exons. Lines with arrows indicate approximate locations of Abi1/Hssh3bp1 structural domains and identified binding regions for listed proteins or their domains. WAVE-BD, WAVE domain-binding region; , HHR, homeo-domain homologous region; Abl-SH3-SH2, c-Abl kinase SH3-SH2 domain-binding region PXXP-, Ser- or Pro-rich, sequences rich in SH3 domain-binding consensus PXXP, serine or proline residues, respectively; SH3, Src homology 3 domain. Indicated binding sites for: Spectrin-SH3, Spectrin Src Homology 3 domain; Eps8-SH3, Eps8 Src Homology 3 domain; p85, p85 regulatory subunit of phosphoinositide-3 kinase; p85-SH2, p85-Src Homology 2 domain; Abl-PRL, c-Abl kinase proline-rich region, N-WASP and mDiaLNCaP Δex6, indicates exon 6 deletion; ATG, start codon. ( b ) DNA sequence chromatograms demonstrating recurring ABI1 mutations. Chromatograms from tumor tissue and tissue adjacent to tumor or archived lymph node biopsy are shown. The text above chromatograms indicates location and primary consequence of mutations. Left panel demonstrates A363S and A363D mutations; middle panel, deletion following codon G331 (#G331); right panel demonstrates complete deletion of exon 10.

Article Snippet: For evaluating protein levels, the following antibodies were used: Abi1 (clone 1B9, MBL International, Woburn, MA, USA); Abi2 (p-20, #sc-20327, Santa Cruz Biotechnology); E-Cadherin (Cat. No. 4065) and phospho-Akt S473 (Cat. No. #9271) (Cell Signaling Technology, Inc., Danvers, MA, USA); B-catenin (Cat. No. 610154, BD Biosciences, San Jose, CA, USA); Ki-67 (Cat. No. VP-K451, Vector Laboratories); WAVE2 (H-110, #sc-33548; #07-410, Santa Cruz Biotechnology; rabbit polyclonal (#1735) and NAP1 were kind gifts from Theresia Stradal).

Techniques: Mutagenesis, Binding Assay, Sequencing

Journal: Oncogenesis

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

doi: 10.1038/oncsis.2012.28

Figure Lengend Snippet: ABI1 mutations in primary prostate tumors

Article Snippet: For evaluating protein levels, the following antibodies were used: Abi1 (clone 1B9, MBL International, Woburn, MA, USA); Abi2 (p-20, #sc-20327, Santa Cruz Biotechnology); E-Cadherin (Cat. No. 4065) and phospho-Akt S473 (Cat. No. #9271) (Cell Signaling Technology, Inc., Danvers, MA, USA); B-catenin (Cat. No. 610154, BD Biosciences, San Jose, CA, USA); Ki-67 (Cat. No. VP-K451, Vector Laboratories); WAVE2 (H-110, #sc-33548; #07-410, Santa Cruz Biotechnology; rabbit polyclonal (#1735) and NAP1 were kind gifts from Theresia Stradal).

Techniques: Mutagenesis, Functional Assay, Phospho-proteomics

Journal: Oncogenesis

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

doi: 10.1038/oncsis.2012.28

Figure Lengend Snippet: Expression of Abi1 in LNCaP cell line inhibits cellular growth, anchorage-independent growth and proliferation in vitro and in xenograft model. ( a ) Inhibition of cell growth, and ( b ) cell proliferation of LNCAP cells expressing Abi1. LNCaP cells transfected with Abi1 proliferate slower than mock-transfected cells. Cell proliferation was evaluated by 5-bromo-2-deoxyuridine incorporation as described in Materials and methods. The data indicate that the percentage of cells in S phase was significantly lower among cells transfected with Abi1 isoform 2 (Abi1) than among cells transfected with isoform 2-lacking exon 10 (Abi1 ΔEx10), or mock transfected, ( P <0.001, χ 2 ). ( c ) Expression of Abi1 but not the mutant Abi1 ΔEx10 promotes cell-to-cell adhesion. Cell aggregation assay was performed as described in Materials and methods. Left panel , quantification of cell-to-cell contacts; *** P <0.001, n =3. Right panel , representative images from indicated cell lines: cells with three or more contacts are indicated by asterisks. ( d ) Abi1 mutant A363S does not inhibit colony formation as well as wt Abi1. LNCaP cell lines expressing Abi1 or Abi1 containing a known prostate cancer mutation in exon 10 (A363S) were plated in soft agar and evaluated for colony growth 4 weeks later. Numbers of colonies over a defined size (>0.5 cm in images taken from agar plates) were scored positive and counted per region of interest (ROI), n =3. A363S cell line showed enhanced growth vs the Abi1 isoform 2 expressing cell line, ** P <0.01; * P <0.05. ( e ) Abi1 inhibits of tumor growth in LNCaP xenograft assay. Growth of tumors was monitored by prostate-specific antigen (PSA) as described in Materials and methods. Expression of Abi1 inhibits secretion of serum PSA. Some animals did not develop tumors hence no secretion of PSA was observed. Abi1, Abi1 isoform 2 expressing cell line; mock, cell line expressing empty plasmid. Comparison between the cell lines indicates statistical significance between the groups ( n =0.0374, two-way analysis of variance).

Article Snippet: For evaluating protein levels, the following antibodies were used: Abi1 (clone 1B9, MBL International, Woburn, MA, USA); Abi2 (p-20, #sc-20327, Santa Cruz Biotechnology); E-Cadherin (Cat. No. 4065) and phospho-Akt S473 (Cat. No. #9271) (Cell Signaling Technology, Inc., Danvers, MA, USA); B-catenin (Cat. No. 610154, BD Biosciences, San Jose, CA, USA); Ki-67 (Cat. No. VP-K451, Vector Laboratories); WAVE2 (H-110, #sc-33548; #07-410, Santa Cruz Biotechnology; rabbit polyclonal (#1735) and NAP1 were kind gifts from Theresia Stradal).

Techniques: Expressing, In Vitro, Inhibition, Transfection, Mutagenesis, Xenograft Assay, Plasmid Preparation, Comparison

Journal: Oncogenesis

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

doi: 10.1038/oncsis.2012.28

Figure Lengend Snippet: The onset of histopathological changes in Abi1 KO mice at 8 months. ( a ) Confirmation of Abi1 gene disruption in mouse prostate. DNA was isolated from Abi1 (fl/fl) mouse prostates (anterior and dorsal lobes) positive for the Cre recombinase-expressing allele (+) or with no Cre presence (−), as shown in the indicated genotypes. PCR using nested primers encompassing the recombined DNA region was carried out as described in Materials and methods. Positive bands were detected only in samples with Cre expression. As controls-immortalized Abl floxed MEF cells were used, parental, that is, of the genotype Abi1 (fl/fl) or upon Cre-mediated disruption of ABI1 gene (clone names above the panel). The fidelity of the Cre-mediated recombination was confirmed by DNA sequencing of PCR fragments. ( b ) The earliest time of observation for mPIN in mice-lacking Abi1, that is, of the genotype Abi1(fl/fl);PBCre(+), is 8 months. Comparison of changes noted in the anterior and dorsal lobes of 8-month-old Abi1(fl/fl);PBCre(+) mice vs age-matched Cre (−) and background control strain mice. Abi1(fl/fl);PBCre(+) mice feature hyperplasia/mPIN changes, with multiple glands lined by crowded epithelial cells, as well as disorderly piling up of cells in the more severely affected areas. Atypical nuclear changes (best visualized in the inserts), including karyomegaly and hyperchromasia, are also present in many glands. Glands from both the Cre (−) and control strain mice are unremarkable. Number of mice evaluated per genotype at 8 months, n >5.

Article Snippet: For evaluating protein levels, the following antibodies were used: Abi1 (clone 1B9, MBL International, Woburn, MA, USA); Abi2 (p-20, #sc-20327, Santa Cruz Biotechnology); E-Cadherin (Cat. No. 4065) and phospho-Akt S473 (Cat. No. #9271) (Cell Signaling Technology, Inc., Danvers, MA, USA); B-catenin (Cat. No. 610154, BD Biosciences, San Jose, CA, USA); Ki-67 (Cat. No. VP-K451, Vector Laboratories); WAVE2 (H-110, #sc-33548; #07-410, Santa Cruz Biotechnology; rabbit polyclonal (#1735) and NAP1 were kind gifts from Theresia Stradal).

Techniques: Disruption, Isolation, Expressing, DNA Sequencing, Comparison, Control

Journal: Oncogenesis

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

doi: 10.1038/oncsis.2012.28

Figure Lengend Snippet: Disruption of ABI1 does not lead to invasive adenocarcinoma in up to 12-month-old mice. ( A ) Summary of observations at 4–12 months of mice lacking both Abi1 alleles, Abi1(fl/fl);PBCre(+). (a–d) At 4 months of age, all prostatic lobes from the Abi1 KO mice appeared normal. (e–h) At 8 months of age, the anterior (e) and dorsal (f) lobes of Abi1 KO mice featured multiple gland profiles lined by crowded cells, many of which had enlarged hyperchromatic nuclei. In several affected areas, cells were piled up in a disorderly fashion (consistent with PIN). Lateral (g) and ventral (h) lobes were unremarkable. (i–l) At 10 months of age, a few glands within the lateral (k) lobes are also affected. Ventral (l) lobes are virtually unaffected. (m–p) At 12 months of age, all lobes of Abi1 KO mice demonstrated some degree of hyperplasia/mPIN changes. These changes were most prominent in the anterior lobe (m), in which the epithelium in the region shown here demonstrated a high degree of cellular atypia. All panels represent hematoxylin and eosin staining (h and e). At least four mice were evaluated per each time point; all mice were positive for histopathological changes at 10 and 12 months. ( B ) Nuclear atypia in Abi1 KO prostate. Enlarged selected panels from ( A ) to indicate nuclear atypia (arrows).

Article Snippet: For evaluating protein levels, the following antibodies were used: Abi1 (clone 1B9, MBL International, Woburn, MA, USA); Abi2 (p-20, #sc-20327, Santa Cruz Biotechnology); E-Cadherin (Cat. No. 4065) and phospho-Akt S473 (Cat. No. #9271) (Cell Signaling Technology, Inc., Danvers, MA, USA); B-catenin (Cat. No. 610154, BD Biosciences, San Jose, CA, USA); Ki-67 (Cat. No. VP-K451, Vector Laboratories); WAVE2 (H-110, #sc-33548; #07-410, Santa Cruz Biotechnology; rabbit polyclonal (#1735) and NAP1 were kind gifts from Theresia Stradal).

Techniques: Disruption, Staining

Journal: Oncogenesis

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

doi: 10.1038/oncsis.2012.28

Figure Lengend Snippet: Prostate tissue lacking Abi1 exhibit enhanced proliferation and loss of cellular adhesion markers downstream of WAVE2 complex downregulation. ( a ) Cells and prostate tissues lacking Abi1 exhibit downregulation of WAVE2 complex components but upregulation of Abi2 as indicated by western blotting analysis. Examination of WAVE2 complex integral proteins as indicated in Abi1 KO MEF cells ( a ); and in prostate tissue ( b ). Please note co-incidental downregulation of E-cadherin in prostate tissue and upregulation of Abi2 evident at 12 months in Abi1 KO MEF cells. Anterior prostates were analyzed at 10 and 12 months, ‘−' indicates absence of probasin Cre recombinase transgene and ‘+' indicates its presence in Abi1 floxed mice. ( b ) Enhanced cellular proliferation in prostate tissue lacking Abi1 correlates with enhanced Abi2 levels in prostate tissue. Top: immunostaining of Abi1 KO prostate tissues with Abi2 antibody. Staining with Abi2 antibody (P20, Santa Cruz Biotechnology) reveals striking upregulation of Abi2 levels in Abi1 KO lesions. Middle: enhanced proliferation as indicated by positive staining with Ki67. Bottom: immunostaining with phospho-Akt S473 antibody indicating staining in the PIN lesion (circle). Right panels, prostate tissue from Abi1 lacking (Abi1(fl/fl);PBCre(+)); left panels, prostate tissue from with Abi1 expression (Abi1(fl/fl);PBCre(+)). Staining of tissue was performed as described in Materials and methods.

Article Snippet: For evaluating protein levels, the following antibodies were used: Abi1 (clone 1B9, MBL International, Woburn, MA, USA); Abi2 (p-20, #sc-20327, Santa Cruz Biotechnology); E-Cadherin (Cat. No. 4065) and phospho-Akt S473 (Cat. No. #9271) (Cell Signaling Technology, Inc., Danvers, MA, USA); B-catenin (Cat. No. 610154, BD Biosciences, San Jose, CA, USA); Ki-67 (Cat. No. VP-K451, Vector Laboratories); WAVE2 (H-110, #sc-33548; #07-410, Santa Cruz Biotechnology; rabbit polyclonal (#1735) and NAP1 were kind gifts from Theresia Stradal).

Techniques: Western Blot, Immunostaining, Staining, Expressing

Journal: Oncogenesis

Article Title: Disruption of Abi1/Hssh3bp1 expression induces prostatic intraepithelial neoplasia in the conditional Abi1/Hssh3bp1 KO mice

doi: 10.1038/oncsis.2012.28

Figure Lengend Snippet: Enhanced activation of Akt and colony formation of cells lacking Abi1. ( a , b ) MEF cells lacking Abi1 exhibit enhanced sensitivity to activation of phospho-Akt downstream from EGFR receptor. Cells were starved overnight and subsequently incubated with epidermal growth factor (10 ng/ml) for indicated amounts of time. ( a ) Cell lysates were prepared as described in Materials and methods, and proteins were analyzed by western blotting with indicated antibodies. ( b ) Quantification of phospho-Akt S473 levels indicate significant increase of p-Akt S473 signal of the Abi1 KO clone #3–11 at 1 min and 5 min ( P <0.01, two-way analysis of variance); clone #3–6 demonstrated enhanced response vs control #3 cell line at 1 min ( P <0.05, t -test). ( c , d ) Disruption of Abi1 expression promotes colony formation in soft agar. Syngeneic MEF cells containing or lacking Abi1 were incubated in soft agar as indicated in Materials and methods, and total numbers of colonies were scored. Cell lines lacking the Abi1 gene exhibit enhanced colony formation ( P <0.001, #3–6; and P <0.01, #3–11) vs control cell line #3 ( c ). Representative images of soft agar colonies from two independent experiments ( d ). #3 represents Abi1 floxed cell line; #3–6 and #3–11 represent Abi1 KO cell lines.

Article Snippet: For evaluating protein levels, the following antibodies were used: Abi1 (clone 1B9, MBL International, Woburn, MA, USA); Abi2 (p-20, #sc-20327, Santa Cruz Biotechnology); E-Cadherin (Cat. No. 4065) and phospho-Akt S473 (Cat. No. #9271) (Cell Signaling Technology, Inc., Danvers, MA, USA); B-catenin (Cat. No. 610154, BD Biosciences, San Jose, CA, USA); Ki-67 (Cat. No. VP-K451, Vector Laboratories); WAVE2 (H-110, #sc-33548; #07-410, Santa Cruz Biotechnology; rabbit polyclonal (#1735) and NAP1 were kind gifts from Theresia Stradal).

Techniques: Activation Assay, Incubation, Western Blot, Control, Disruption, Expressing

Journal: Oncotarget

Article Title: Reciprocal regulation of Abl kinase by Crk Y251 and Abi1 controls invasive phenotypes in glioblastoma

doi:

Figure Lengend Snippet: In vitro kinase assay performed with bacterially purified WtCrk and Abi1-Iso2 and Abl-1b purified from SF9 cells. 10nM Abl-1b was incubated with 1uM Crk alone or in presence of increase concentrations of Abi1-Iso2 with ATP at 25°C for 10mins. A. Crk mediated Abl transactivation was significantly suppressed in samples containing Abi1-Iso2 as shown by Abl pY245 blots. B. In another variation, Abi1 alone downregulates Abl activation alone and in presence of Crk in dose- dependent manner as indicated by Abl pY412 blot. C. - D. In vitro kinase assay to assess Abi1 SH3 domain on Abl kinase activity: Abl-1b and Crk were co-incubated with either C. Wt SH3 domain or D. mutant SH3 domain of Abi1-Iso2 in increasing amounts. In a dose-dependent manner Abi1 Wt-SH3 domain significantly suppresses Abl-1b-mediated CrkY221 phosphorylation (C), whereas mutant SH3 domain looses the capacity to suppress aforementioned CrkY221 phosphorylation (D). E. Competitive kinase assay: Abl kinase was co-incubated either with Crk alone or Crk and 2μM Abi1-Iso2 SH3 domain in kinase buffer. After 30min, Abl kinase activity was assessed by CrkY221 phosphorylation. Data are represented as mean ± SEM and n = 3 ( P < 0.05).

Article Snippet: Western blot analyses were performed using EGFR, phospho-Y221 CrkII, previously described phospho- Crk Y251 (CST), anti-pY245 Abl, and anti-pY412 Abl (Cell Signaling Technology Inc.), phosphotyrosine antibody PY99, GST (Santa Cruz Biotechnology Inc.), Abl Ab-3 (Calbiochem), and Abi1 1B9 antibodies.

Techniques: In Vitro, Kinase Assay, Purification, Incubation, Activation Assay, Activity Assay, Mutagenesis

Journal: Oncotarget

Article Title: Reciprocal regulation of Abl kinase by Crk Y251 and Abi1 controls invasive phenotypes in glioblastoma

doi:

Figure Lengend Snippet: A. HEK293T cells were transfected with plasmids encoding WtAbl-1b or constitutively active, partially open conformation Abl PP alone or co-transfected with increasing amount of Abi1-Iso2 plasmid DNA. Abl PP constitutive activity is downregulated to markedly low levels (Abl pY412 blot) that correlates with Abi1-Iso2 expression level (HA blot) in cells. Abi1 Y213 phosphorylation increases with increasing amount of Abi1 in these cells. Endogenous Crk Y251 phosphorylation and general phosphotyrosine levels (PY99) that are mediated by Abl PP are downregulated by Abi1-Iso2. B. Crk co-transfected with Abl PP in the similar experimental design superactivates Abl PP as indicated by PY99 blot. C. In vitro kinase assay using Abl PP and Abi1-Iso2: Abl PP was co-incubated with Abi1-Iso2 in kinase buffer for 30min, reactions were boiled and immunoblotted for Abl pY412. D. Schematic representation of effect of Crk and Abi1-Iso2 on differential regulation of Abl PP activity.

Article Snippet: Western blot analyses were performed using EGFR, phospho-Y221 CrkII, previously described phospho- Crk Y251 (CST), anti-pY245 Abl, and anti-pY412 Abl (Cell Signaling Technology Inc.), phosphotyrosine antibody PY99, GST (Santa Cruz Biotechnology Inc.), Abl Ab-3 (Calbiochem), and Abi1 1B9 antibodies.

Techniques: Transfection, Plasmid Preparation, Activity Assay, Expressing, In Vitro, Kinase Assay, Incubation

Journal: Oncotarget

Article Title: Reciprocal regulation of Abl kinase by Crk Y251 and Abi1 controls invasive phenotypes in glioblastoma

doi:

Figure Lengend Snippet: A. Upon stimulation with hEGF, Crk and Abl kinase are phosphorylated within 1min in U138 and HS683 GBM cells. B. Canonical Crk signaling is seen by CrkY221 phosphorylation by hEGF in GBM cell lines. C. Schematic figure of Crk indicating Tyr 221, 239 and 251 in context of its modular domains. D. Parental HS683 cells were pretreated with or without 10uM Imatinib and stimulated with or without hEGF after which detergent lysates were made and immunoblotted with Abl pY245 and Crk pY251 antibodies. E. Western blot analysis performed to assess the stable expression of MSCV-EYFP (Empty vector), MSCV-EYFP-Crk and MSCV-EYFP-CrkY251F in HS683 cells. F. Wound healing assay: Cell migration of the HS683 stable cells was assessed by imaging the rate of wound healing by serum starved, hEGF stimulated cells in triplicate samples at 6 and 12 hours time points. G. Percentage wound healing was calculated in all cell lines and data were represented as mean ± SEM ( n = 3) ( P < 0.05). H. Cell invasion by Matrigel-coated Boyden chambers: Post-serum starvations for 12-16hrs, 10,000 cells were seeded in serum free media. 10% FBS containing media was used as chemoattractant in the lower chamber. Cell were fixed, stained and imaged as described (see methods) and quantified to represent data in fold change in cell invasion (left panel). I. Cell invasion using real-time xCELLigence-based assay: Overnight serum starved cells were seeded in serum free media in triplicates in 40000 cells/well. Cell invasion was assayed every 15min for indicated time using 10% FBS containing media as chemoattractant ( P < 0.05). J. xCELLigence assay to test the effect of Imatinib treatment on cell invasion of Hs683 cells. K. Distinct morphological features by cells expressing vector (EYFP), Crk, or CrkY251F mutant. L. Micrographs of cell spreading on fibronectin coated dishes: 1000 respective cells were seeded on fibronectin pre-coated dishes (10ug/ml) and images were taken from multiple fields (3 fields/cell line shown) 30min later using 40X objective of phase contrast microscope. M. Real-time xCELLigence-based cell spreading assay on fibronectin-coated CIM-16 plates seeded with 10,000 vector, Crk, CrkY251F expressing HS683 cells using 10%FBS as chemoattractant. N. CrkY251 phosphorylation by fibronectin in stable cell lines: The stable cell lines that were seeded on fibronectin dishes for 30min were harvested, lysed and immunoblotted for endogenous and EYFP- CrkY251 phosphorylation status. O. Tabular summary of the CrkY251-mediated biological phenotypes observed. See also .

Article Snippet: Western blot analyses were performed using EGFR, phospho-Y221 CrkII, previously described phospho- Crk Y251 (CST), anti-pY245 Abl, and anti-pY412 Abl (Cell Signaling Technology Inc.), phosphotyrosine antibody PY99, GST (Santa Cruz Biotechnology Inc.), Abl Ab-3 (Calbiochem), and Abi1 1B9 antibodies.

Techniques: Western Blot, Expressing, Plasmid Preparation, Wound Healing Assay, Migration, Imaging, Staining, Mutagenesis, Microscopy, Stable Transfection

Journal: Oncotarget

Article Title: Reciprocal regulation of Abl kinase by Crk Y251 and Abi1 controls invasive phenotypes in glioblastoma

doi:

Figure Lengend Snippet: In vitro kinase assay performed with bacterially purified WtCrk and Abi1-Iso2 and Abl-1b purified from SF9 cells. 10nM Abl-1b was incubated with 1uM Crk alone or in presence of increase concentrations of Abi1-Iso2 with ATP at 25°C for 10mins. A. Crk mediated Abl transactivation was significantly suppressed in samples containing Abi1-Iso2 as shown by Abl pY245 blots. B. In another variation, Abi1 alone downregulates Abl activation alone and in presence of Crk in dose- dependent manner as indicated by Abl pY412 blot. C. - D. In vitro kinase assay to assess Abi1 SH3 domain on Abl kinase activity: Abl-1b and Crk were co-incubated with either C. Wt SH3 domain or D. mutant SH3 domain of Abi1-Iso2 in increasing amounts. In a dose-dependent manner Abi1 Wt-SH3 domain significantly suppresses Abl-1b-mediated CrkY221 phosphorylation (C), whereas mutant SH3 domain looses the capacity to suppress aforementioned CrkY221 phosphorylation (D). E. Competitive kinase assay: Abl kinase was co-incubated either with Crk alone or Crk and 2μM Abi1-Iso2 SH3 domain in kinase buffer. After 30min, Abl kinase activity was assessed by CrkY221 phosphorylation. Data are represented as mean ± SEM and n = 3 ( P < 0.05).

Article Snippet: Western blot analyses were performed using EGFR, phospho-Y221 CrkII, previously described phospho- Crk Y251 (CST), anti-pY245 Abl, and anti-pY412 Abl (Cell Signaling Technology Inc.), phosphotyrosine antibody PY99, GST (Santa Cruz Biotechnology Inc.), Abl Ab-3 (Calbiochem), and Abi1 1B9 antibodies.

Techniques: In Vitro, Kinase Assay, Purification, Incubation, Activation Assay, Activity Assay, Mutagenesis

Journal: bioRxiv

Article Title: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

doi: 10.1101/2020.05.11.083030

Figure Lengend Snippet: (A) Still images from live cell imaging showing NHSL1-EGFP co-expressed with mScarlet-I-tagged Abi1 and (B) Nap1 in B16-F1 cells. Representative images shown from three independent experiments. Scale bar represent 20 µm. Inset is a magnified view of the white box. Scale bar in inset (A) applies to both insets (A,B): 1.25 μm. See also related video S6. (C-E) Endogenous NHSL1 co-localises with Abi1 (C, NHSL1 pAb) Scar/WAVE1 (D, NHSL1 pAb ) , and Scar/WAVE2 (E, NHSL1 mAb ) at the very edge of lamellipodia in B16-F1 mouse melanoma cells. Scale bar in (C) applies also to (D,E): 20 µm. Inset represents a magnified view of the white box. Scale bar in inset in (C) applies also to inset for (D,E): 20 μm. Representative images shown from three independent experiments. See Fig. S8 for line scans of co-localisations in (C) and (E).

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Abi2 (Epitomics, 3189-1), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), ARPC2 (07-227-I-100UG, Millipore).

Techniques: Live Cell Imaging

Journal: bioRxiv

Article Title: Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration

doi: 10.1101/2020.05.11.083030

Figure Lengend Snippet: (A) Example B16-F1 cell shown indicating definition of length and width used in (B; S17A-D): First frame of a movie of LifeAct-EGFP expressing B16-F1 cells to show how length, width and area was quantified: red line indicates area of the lamellipodium. The length was quantified using the Fiji plugin “measure_ROI” ( http://www.optinav.info/Measure-Roi.htm:Measure_Roi_Curve.java ) which measures the length of curved objects (see materials and methods for details). For the width, the line tool in Fiji was used to draw lines (blue lines in (A)) at 90 degree angle to the leading edge to measure width of lamellipodium at 10 roughly equal distance points along the leading edge and the mean of these measurements was used. Scale bar: 20 μm. (B) Quantification of the ratio lamellipodia length/total cell area, One-way ANOVA, Dunnett’s multiple comparisons test; ****, p< 0.0001; **, p=0.0059; F(2,42)=10.84 (C) Quantification of total cellular F-actin (Alexa568 phalloidin) intensity/area ratio in wild type B16-F1 cells expressing Myc alone as control (black circles) or CRISPR 2 cells expressing either the NHSL1 mutant in the Scar/WAVE complex binding sites (NHSL1 SW Mut, blue diamonds) or NHSL1 (NHSL1 WT, red crosses) or Myc alone as control (pink squares) plated on laminin. Results are mean ± SEM (error bars) from four independent biological experiments. One-way ANOVA: p=0.0009; Kruskal Wallis statistics 16.44; ***, p=0.0003; **, p=0.0083; ns, p=0.1518; Outliers were removed using the ROUT method (Q=1%) (n analysed/ n outliers = n o removed); WT control: n=67 cells/ n o =0; NHSL1 CRISPR2: n=71 cells/ n o =3; NHSL1 CRISPR2+Myc-NHSL1 SW Mut Rescue: n=76 cells/ n o =5; NHSL1 CRISPR2+Rescue Myc-NHSL1 WT: n=82 cells/ n o =4. (D) Quantification of relative lamellipodial Arp2/3 intensity: Arp2/3 intensity in the lamellipodium was normalized cell-by-cell against Arp2/3 intensity of the whole cell. Kruskal-Wallis test (p=0.0514) and Dunn’s multiple comparisons test: * p=0.0353; ns p>0.9999; in wild-type (n=40 cells, control), NHSL1 CRISPR 2 (n=38 cells) and NHSL1 CRISPR 21 (n=33 cells) B16-F1 cells plated on laminin and stained with anti-ARPC2 (subunit of Arp2/3 complex) antibodies; Results are mean ± SEM (error bars), three independent biological repeats. (E) Quantification of lamellipodial F-actin (Alexa488 phalloidin) intensity/area ratio in B16-F1 wild-type control cells or NHSL1 CRISPR 2 and NHSL1 CRISPR 21 B16-F1 cells plated on laminin. Results are mean ± SEM (error bars), five independent biological repeats, n= control (61), CRISPR 2 (61), CRISPR 21 (43) cells; One-way ANOVA: p=0.0127; F(2,162)=4.486; and Dunnett’s multiple comparisons test: **, p=0.0099. (F) Quantification of lamellipodial F-actin (LifeAct-EGFP) intensity/area ratio in B16-F1 cells transfected with a tri-cistronic plasmid to express Myc-tagged NHSL1 (wild type or Scar/WAVE binding mutant), puromycin resistance and LifeAct-EGFP or empty plasmid (Myc-IRES-Puro-T2A-LifeAct-EGFP) after selection with puromycin plated on laminin. Results are mean ± SEM (error bars), four independent experiments, n= control (14), WT (12), NHSL1-SW mut (13) cells. One-way ANOVA: p=0.0074; F(2,36)=5.636; and Dunnett’s multiple comparisons test: **, p=0.0036; ns p=0.1810. Source data are provided as a Source Data file.

Article Snippet: Commercial primary antibodies: EGFP (Roche), Myc (9E10, Sigma), MBP (New England Biolabs), Abi1 (MBL, clone 1B9, D147-3), Abi2 (Epitomics, 3189-1), Scar/WAVE1 (BD 612276), Scar/WAVE2 rabbit mAb (D2C8, CST 3659), ARPC2 (07-227-I-100UG, Millipore).

Techniques: Expressing, CRISPR, Mutagenesis, Binding Assay, Staining, Transfection, Plasmid Preparation, Selection

Journal: Cell communication and signaling : CCS

Article Title: Abi1 loss drives prostate tumorigenesis through activation of EMT and non-canonical WNT signaling.

doi: 10.1186/s12964-019-0410-y

Figure Lengend Snippet: Fig. 6 Differential gene expression pattern is correlated with EMT pathways and WNT signaling. The DEGs determined by RNA sequencing demonstrated (a) upregulation of EMT pathways and Wnt signaling. Individual genes, fold changes and p-values are listed. Right panel shows representative western blots of several EMT markers and different integrin proteins validate the RNA sequencing findings. (b) Western blots showing upregulation of STAT3 phosphorylation (Y705 and S727) and FYN activation. (c) ABI1 interacts with STAT3 and FYN in RWPE-1 cells. Western blotting results indicating that FYN (top panel) and STAT3 (middle panel) co-immunoprecipitated with Abi1. Input, RWPE-1 cell lysate; Flow-Through, unbound fraction of the lysate; IgG, control immunoprecipitation lacking anti-Abi1 antibody; IP, immunoprecipitation including the anti-Abi1 antibody. Asterisk in top panel indicates the corresponding bands in fractions. (d-f) ABI1 loss promotes nuclear localization of activated STAT3 as determined by pY705 antibody. (d) Western blotting analysis of p-STAT3 Y705 expression in cytoplasmic (left panel) or, nuclear fraction (right panel) of RWPE ABI1 KO, control and ABI1-rescued clones. (e) Immunofluorescence analysis of p-STAT3 Y705 levels in RWPE ABI1 KO clones. (f) Quantification of nuclear p-STAT3 Y705 levels, n = 3, p < 0.001. (g) An example of inverse correlation of ABI1 and pSTAT3 Y705 expression levels in a prostate tumor. Serial tumor sections were immunostained with antibodies to either ABI1 (left panel), or pSTAT3 Y705 (right panel). Arrows depict two example areas of correlation of low ABI1 and high pSTAT. (h) Schematic depicting the proposed mechanism of how Abi1 loss promotes the invasive potential of prostate epithelial cells. Center, ABI1 (ABI1) is critical for cell junction maintenance through WAVE complex- mediated actin polymerization; disruption of ABI1 leads to loss of WAVE complex (WAVE complex) stability, resulting in lower levels of E-cadherin (CDH1) at cell-cell contacts and downregulation of cell-cell adhesion. Right, ABI1 pY421 binds with high affinity to FYN-SH2 domain. Disruption of ABI1 leads to constitutive activation of the SRC family kinase FYN (FYN) to stimulate STAT3 activation and promote its nuclear localization. Nuclear STAT3 promotes transcription of the epithelial-to-mesenchymal transition (EMT) gene program, including extracellular matrix metalloproteinases that promote migration through matrix degradation in the absence of proper cell-cell adhesion. Left, In the absence of ABI1, activated FYN acts on FAK kinase (FAK) to promote integrin signaling, which also supports cell migratory activity. Activated FAK may also directly bind and activate pSTAT3, thus providing another possibility for EMT pathway activation

Article Snippet: ABI1 was immunoprecipitated with MBL Mab 1B9: For Western blotting we used Abi1 CST 39444 (bottom panel), STAT3 CST 30835 (middle panel); FYN immunoreactivity was detected with BioRad VPA00019. (B) Recombinant ABI1 interacts with STAT3 (middle panels), and co-immunoprecipitated active phospho-SRC activity as indicated by pan phospho-SRC family kinase antibody to pY416 (CST 2101), lower panels.

Techniques: Gene Expression, RNA Sequencing, Western Blot, Phospho-proteomics, Activation Assay, Immunoprecipitation, Control, Expressing, Clone Assay, Immunofluorescence, Disruption, Migration, Activity Assay