antibodies anti abi1  (Proteintech)

Journal: Translational oncology

Article Title: CBLC promotes the development of colorectal cancer by promoting ABI1 degradation to activate the ERK signaling pathway.

doi: 10.1016/j.tranon.2024.101992

Figure Lengend Snippet: Fig. 6. CBLC promoted CRC cell growth and lung metastasis in vivo. (A) Xenograft tumor formed by subcutaneous injection of infected CRC cells. (Scale bars, 1 cm). (B-C) IHC staining showed the expression of CBLC and Ki67 in the xenograft tumors. (The arrows represented positive cells; Scale bars, 50 µm). (D) Photographs showed representative mouse lungs (Scale bars, 1 cm), and the number of pulmonary nodules was quantified. (E) H&E staining was used to analyze tumor cell metastasis in lung tissues. (F) The levels of ABI1, ERK and p-ERK (Thr202/Tyr204) in tumor tissues were detected by western blotting. (Scale bars, 100 µm). Mean ± SD. p < 0.0001.

Article Snippet: The antibodies used were as follows: CBLC antibody (1:1000, SAB2900314, Sigma); ABI1 antibody (1:500, 66,609–1-Ig, Proteintech) and ubiquitin antibody (1: 5000, 10,201–2-AP, Proteintech).

Techniques: In Vivo, Injection, Infection, Immunohistochemistry, Expressing, Staining, Western Blot

Journal: Translational oncology

Article Title: CBLC promotes the development of colorectal cancer by promoting ABI1 degradation to activate the ERK signaling pathway.

doi: 10.1016/j.tranon.2024.101992

Figure Lengend Snippet: Fig. 8. CBLC led to the ubiquitination and degradation of ABI1. (A) Double immunofluorescence staining was used to analyze the co-localization of CBLC and ABI1 in HCT116 and LOVO cells. (The rectangular box magnified the co-localization of these two proteins; Scale bars, 50 µm). (B-C) Co-immunoprecipitation and western blotting assays were applied to determine binding of CBLC and ABI1 and ABI1 ubiquitination levels. (D) ABI1 expression was measured by western blotting after overexpression or knockdown of CBLC in HCT116 cells.

Article Snippet: The antibodies used were as follows: CBLC antibody (1:1000, SAB2900314, Sigma); ABI1 antibody (1:500, 66,609–1-Ig, Proteintech) and ubiquitin antibody (1: 5000, 10,201–2-AP, Proteintech).

Techniques: Ubiquitin Proteomics, Double Immunofluorescence Staining, Immunoprecipitation, Western Blot, Binding Assay, Expressing, Over Expression, Knockdown

Journal: Translational oncology

Article Title: CBLC promotes the development of colorectal cancer by promoting ABI1 degradation to activate the ERK signaling pathway.

doi: 10.1016/j.tranon.2024.101992

Figure Lengend Snippet: Fig. 9. ABI1 overexpression abolished the effects of CBLC on the tumorigenesis of CRC. (A) ABI1 overexpression lentiviral vector was constructed and infected into HCT116 cells. The overexpression efficiency of ABI1 was examined by qPCR and western blotting. (B-C) Cell proliferation and colony formation were detected by CCK-8 and colony formation assays. (D) Cell cycle progression was analyzed by flow cytometry. (E-F) The migratory and invasive abilities of CRC cells were evaluated by scratch (Scale bars, 200 µm) and transwell (Scale bars, 100 µm) assays. (G) The levels of ERK and phosphorylated ERK (Thr202/Tyr204) were measured by western blotting. Mean ± SD. p < 0.05; p < 0.01; p < 0.0001.

Article Snippet: The antibodies used were as follows: CBLC antibody (1:1000, SAB2900314, Sigma); ABI1 antibody (1:500, 66,609–1-Ig, Proteintech) and ubiquitin antibody (1: 5000, 10,201–2-AP, Proteintech).

Techniques: Over Expression, Plasmid Preparation, Construct, Infection, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Translational oncology

Article Title: CBLC promotes the development of colorectal cancer by promoting ABI1 degradation to activate the ERK signaling pathway.

doi: 10.1016/j.tranon.2024.101992

Figure Lengend Snippet: Fig. 10. Schematic diagram for the role of CBLC in CRC. CBLC acts as a tumor promoter in CRC through triggering the ubiquitination and degradation of ABI1 to activate the ERK signaling pathway. DNA: https://doi.org/10.5281/zenod o.4072322.

Article Snippet: The antibodies used were as follows: CBLC antibody (1:1000, SAB2900314, Sigma); ABI1 antibody (1:500, 66,609–1-Ig, Proteintech) and ubiquitin antibody (1: 5000, 10,201–2-AP, Proteintech).

Techniques: Ubiquitin Proteomics

Journal: bioRxiv

Article Title: ABI1 regulates transcriptional activity of Androgen Receptor by novel DNA and AR binding mechanism

doi: 10.1101/2023.05.26.542350

Figure Lengend Snippet: A , Analysis of ABI1 expression (IHC with ABI1 antibody, quantified by digital imaging) in VPC TMA cohort of neoadjuvant hormone treatment (NHT) of PCa tumors. Numbers within bars show the total number of cases analyzed for each group, *, p<0.05; **, p<0.01; ****, p<0.0001. B , representative IHC images from TMA analysis in panel A . C-D , Correlation of ABI1 and AR mRNA expression in patient samples, in TCGA samples, PanCancer Atlas (n=494) C ; or in Firehose/PRAD cohort, (n=500) D . E , ABI1 immunoexpression in TMA samples of LuCaP PDX cell lines, letters in left upper corners of images define position on TMA slides (HS, LuCaP 35/81/136; CRPC, LuCaP 35CR, 81CR, 136CR; NEPC, LuCaP, 77CR/86.2/93/145.1/145.2). F Quantification of ABI1 expression in E , n=3,** p<0.01. G , Abi1 immunoexpression in mouse prostate epithelium was decreased after 4 weeks of castration compared to non-castrated controls quantified in H , n=3, **p<.0098. Abbreviations: HS, Hormone sensitive; CRPC, castrate resistant prostate cancer; TURP, transurethral resection of the prostate; NEPC, neuroendocrine prostate cancer; IB, immunoblot detection of indicated antibody target .

Article Snippet: For ChIP analysis we used anti-Abi1 antibody (MBL International, cat# D147-3, Lot# 034), AR(N20, SCBT, sc-816).

Techniques: Expressing, Imaging, Western Blot

Journal: bioRxiv

Article Title: ABI1 regulates transcriptional activity of Androgen Receptor by novel DNA and AR binding mechanism

doi: 10.1101/2023.05.26.542350

Figure Lengend Snippet: A. Expression of ABI1 is enhanced with synthetic androgen, R1881 (1 nM), but downregulated with AR inhibitor Enzalutamide (Enza) (5 μM). B . Quantification of A , DMSO control versus R1881 (**p<0.007) and control versus R1881 + Enza (*p<0.02); n=3. C. UCSC genome browser-based diagram depicting AR binding site within ABI1 gene. Black lines indicate binding events at specific locations across chromatin. Red box indicates AR binding locations on ABI1 . D , ChIP-qPCR assays indicates a fold enrichment of AR binding to the 2nd intron binding site within ABI1 upon overexpression of AR and DHT treatment in the LNCaP cells. E , Induction of ABI1 mRNA expression upon AR overexpression, LNCaP-AR vs. LNCaP-empty vector in DHT (1 nM) treated cells. F , Enhanced E-cadherin localization to cell-cell junction as functional readout of ABI1 at 48h and further at 72h (lower panels) compared to control but disrupted in ABI1 KO cell lines (right panels), quantified in G . H . Inhibition of E-cadherin immuno-intensity at cell-cell junctions following Enza treatment alone or following R1881, vs. control (DMSO) (see Supplementary Figure S1B for representative images). Line intensities were quantified from 50 junctions per treatment; n=3; p<0.005.

Article Snippet: For ChIP analysis we used anti-Abi1 antibody (MBL International, cat# D147-3, Lot# 034), AR(N20, SCBT, sc-816).

Techniques: Expressing, Binding Assay, Over Expression, Plasmid Preparation, Functional Assay, Inhibition

Journal: bioRxiv

Article Title: ABI1 regulates transcriptional activity of Androgen Receptor by novel DNA and AR binding mechanism

doi: 10.1101/2023.05.26.542350

Figure Lengend Snippet: A , Protein domain map of AR-FL/V7 and ABI1 with red boxes indicating their respective binding domains. B , Co-IP of ABI1 and AR in naïve VCaP, LAPC4 ( C ), LNCaP ( D ), 22Rv1 ( E ) cell lines indicating the interaction of ABI1 with the full-length AR (AR-FL) (all cell lines), and a truncated version (AR-V7) in 22Rv1 following the treatment with 1 nM R1881. F, Co-IP of ABI1 and AR-FL/ARV7 in a PC3 ABI1 isoform 2 (Iso2-WT) cell line transfected with AR-FL or ARV7. G-H , Co-IP of ABI1 and AR in LNCaP CRISPR ABI1 KO cells transfected with ABI1-WT, (Iso2-WT), or SH3 domain binding mutant (Iso2-W485N) ( G ), or SH3 domain deleted mutant (Iso2-ΔSH3) ( H ) showed decreased or no observable binding to AR vs. WT control, respectively. I. AR-FL/AR-V7 and ABI1 PLA interactions indicated by red color puncta, counterstained with DAPI in LNCaP cell line ( top panel ); 22Rv1 cell line ( middle panel ); positive control ( bottom panel inset ); negative control ( bottom panel ). Cells were incubated in charcoal stripped serum for 5 hours followed by 1 nM R1881 treatment overnight. J. AR-V7 and ABI1 PLA interactions (brown puncta) in primary, neoadjuvant hormone therapy treated, and castrate-resistant prostate cancer patient tumors. K , Immunoblot detection of AR and ABI1 protein expression in subcellular fractions of 22Rv1 cells treated with vehicle control or 1 nM R1881 for 24 hours. Loading controls include GAPDH (cytoplasmic fraction) and PARP (nuclear fraction). L , Co-immunoprecipitation of ABI1 and AR in subcellular fractions of LNCaP cells transfected with the recombinant ABI1 isoform 2. GAPDH, loading control. * Abbreviations: NHT, neoadjuvant hormone therapy; CRPC, castrate resistant prostate cancer; CE, cytoplasmic fraction; NE, nuclear fraction; Cyto, cytoplasmic fraction; Nuclear, nuclear fraction; IgG, immunoglobulin G (negative control); IP, immunoprecipitation and antibody immunoprecipitated for; IB: antibody probe for on immunoblot; AR-FL: AR full length, AR-V7: AR splice variant 7. Red asterisks indicate ABI1 immunoreactive bands .

Article Snippet: For ChIP analysis we used anti-Abi1 antibody (MBL International, cat# D147-3, Lot# 034), AR(N20, SCBT, sc-816).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Transfection, CRISPR, Mutagenesis, Positive Control, Negative Control, Incubation, Western Blot, Expressing, Immunoprecipitation, Recombinant, Variant Assay

Journal: bioRxiv

Article Title: ABI1 regulates transcriptional activity of Androgen Receptor by novel DNA and AR binding mechanism

doi: 10.1101/2023.05.26.542350

Figure Lengend Snippet: A, Immunoblot detection of AR, ABI1, PARP, and GAPDH in subcellular fractions of LNCaP cells: parental, ABI1 KO, ABI1 isoform 2 (Iso2), ABI1 isoform 2 SH3 domain mutant (Iso2-W485N). B , Quantification of AR in A , indicates decreased nuclear localization of AR in ABI1 KO cells vs. cells rescued with ABI1 WT (Iso2) but not with the SH3 binding mutant W485N, (Iso2-W485N). Relative protein expression was quantified from densitometry measurements and normalized to fraction specific loading control either cytoplasmic fraction, GAPDH or nuclear fraction, PARP after subtraction of background signal. % Nuclear accumulation = [Nuclear Fraction/(Cytoplasmic fraction + Nuclear fraction)] * 100; n=3, **p<0.01. C , Transcriptional activity of known AR targets such as KLK3, FKBP5, and novel target gene Wasf1 (Wave1) but not for TMPRSS2 is reduced in cells lacking ABI1 (ABI1 KO) and in cells expressing SH3 binding mutant W485N, (Iso2-W485N). Scatter plots represent ΔΔCq mRNA expression collected using TaqMan Probe qPCR assay for known target genes of AR; n=3, *p<0.05, **p<0.01. D. Distribution of ABI1 peaks over genomic regions (as indicated) from ABI1 ChIP-seq data wherein ABI1 predominantly binds promoter regions suggesting ABI1 may play a role in transcription factor recruitment. E , Top AR-overlapping genes regulated by ABI1. F , VENN Diagram of “KO” represents overlapping peaks of AR ABI1 KO ChIP replicates 1 and 2 and “Rescue” represents overlapping peaks of AR ABI1 Rescue ChIP replicates 1 and 2. Unique peaks identified between samples were, KO=9902 and Rescue=8303 whereas common peaks= 30793. G , AR ChIP-seq data set illustrating 20 genes that have increased (left) or decreased (right) AR chromatin binding identified using genomic regions containing 1 or multiple overlapping Intervals and the associated FDR or “padj” cutoff for significance FDR < 0.001 based on DESeq2 analysis of 50kb. H. De Novo Homer Motif analysis from ABI1 ChIP seq samples indicated log ranked p-values in order of significance using Homer Motif analysis software. I , Hierarchal clustering map from RNAseq data for ABI1-Iso2-WT/KO cell lines (Iso2 rescue, or KO) shows 4 groups of genes positively and negatively regulated by ABI1, in the presence (0 nM) or absence of androgen treatment, R1881 (1nM). ABI1 negatively regulates gene expression of the first seven genes and treatment with AR canonical ligand further induces downregulation when ABI1 is present. J , Pathway enrichment analysis from RNAseq data shows that ABI1 is involved in pathways such as cell adhesion, JAK/STAT, FoxO, transcriptional misregulation in cancer. Labels: green, cytoskeleton/adhesion; yellow, DNA binding; blue, cytosolic component; pink, RNA/splicing factor; orange, tumor suppressor; purple, signaling molecule .

Article Snippet: For ChIP analysis we used anti-Abi1 antibody (MBL International, cat# D147-3, Lot# 034), AR(N20, SCBT, sc-816).

Techniques: Western Blot, Mutagenesis, Binding Assay, Expressing, Activity Assay, ChIP-sequencing, Software

Journal: bioRxiv

Article Title: ABI1 regulates transcriptional activity of Androgen Receptor by novel DNA and AR binding mechanism

doi: 10.1101/2023.05.26.542350

Figure Lengend Snippet: A-B . Recombinant ABI1 (full-length, A or HHR domain, B ) binding to selected double-stranded (ds) DNA probes. Please note similar affinities of ABI1-HHR and the full-length ABI1; genes symbols are indicated above graphs. C , ABI1-HHR binding affinities to HOXB4 dsDNA probe (dsHOXB4) or its mismatched probe (dsHOXB4 mismatched, with 2 non-paring DNA resides in the center); affinities are listed on the right. HHR-WT and HHR-DelEx4, represent wild-type ABI1-HHR or its Exon 4 deleted version, respectively. D-E. RNA-seq and the corresponding clinical data of PCa patients from the TCGA cohort ( https://www.cancer.gov/tcga ) were analyzed for expression of ABI1 Exons 4, 8, and 10; percent of spliced-in (PSI) of ABI1 Exon 4, 8, or 10 are indicated. D , Analysis of ABI1 Exon expression within Gleason groups, or within tumor stages ( E ). F. Kaplan-Meier curves plot indicates disease-free survival of PCa patients by exon inclusion. ABI1 Exon 4 expression demonstrates lower survival of PCa patients. PCa patients were divided into low- and high-groups according to the PSI level. for each ABI1 Exon separately. G-J. Changes in chemical shifts of residues V145 ( G-H ) and K102 ( I-J ) in ABI1-HHR ( G and I ) and ABI1-HHR-DelEx4 ( H and J ) upon gradual addition of HOXB4. Dark blue denotes the free form and black is the bound form, with a color gradient going through grey as the concentration of HOXB4 increases. K. Comparison of Chemical Shift Perturbations (CSPs) of ABI1-HHR (grey) and ABI1-HHR-DelEx4 (black) in presence of HOXB4 at stoichiometry 1:3.75. Blue rectangles define regions encompassing groups of residues with CSPs higher than one standard deviation from the median CSP in ABI1-HHR. L. CSPs reporting on the deletion of residues 155-159 in ABI1-HHR-DelEx4. Red bars identify residues showing CSPs one standard deviation above the median value. Significant CSPs away from the deletion site reveal allosteric responses. Light grey rectangles in ( K ) and ( L ) highlight residues deleted in ABI1-HHR-DelEx4 (155-159). (‡) identify prolines and (†) identify residues whose peaks could not be centered due to overlap in at least one spectrum required for analysis.

Article Snippet: For ChIP analysis we used anti-Abi1 antibody (MBL International, cat# D147-3, Lot# 034), AR(N20, SCBT, sc-816).

Techniques: Recombinant, Binding Assay, RNA Sequencing Assay, Expressing, Concentration Assay, Standard Deviation

Journal: PLoS ONE

Article Title: Differential Regulation of Macropinocytosis by Abi1/Hssh3bp1 Isoforms

doi: 10.1371/journal.pone.0010430

Figure Lengend Snippet: A. Schematic comparison of Abi1 isoform 2 and isoform 3. Comparison of the primary structure of isoform 2 and 3 indicates lack of exon 10 in isoform 3 (Ziemnicka-Kotula et al 1998). Exon 10 encodes a 29 amino acid proline-rich region. Abi1 contains several domains: T-SNARE, homeobox homology region (HHR), PXXP sequence-rich region (PXXP RR), Proline-rich region (PRR), and an SH3 domain. B. Expression of endogenous Abi1 in LNCAP cells. Abil was immunoprecipitated from lysates of the indicated cell lines with monoclonal antibody, 7B6, and blotted with polyclonal antibody, Ab-2. LNCaP indicates LNCAP cell line; PrEC indicates primary prostate cells; IgG indicates a cross-reactive IgG band. C. Ectopic expression of Abi1 isoforms in LNCaP cells. Ha-tagged recombinant Abi1 was immunoprecipitated from cell lysates with monoclonal anti-HA antibody and blotted with the polyclonal anti-HA antibody. Iso-2.1 and Iso-2.2, indicate independent cell lines expressing isoform 2 of Abi1; Iso-3.1 and Iso-3.2 indicate independent cell lines expressing isoform 3 of Abi1; Mock, indicates mock transfected cell line; LNCaP indicates the LNCAP cell line. IgG indicates a cross-reactive IgG band.

Article Snippet: Antibody 1G9 to Abi1 was from MBL International Corporation (Woburn, MA).

Techniques: Sequencing, Expressing, Immunoprecipitation, Recombinant, Transfection

Journal: PLoS ONE

Article Title: Differential Regulation of Macropinocytosis by Abi1/Hssh3bp1 Isoforms

doi: 10.1371/journal.pone.0010430

Figure Lengend Snippet: A. Isoform 2 expressing cell line exhibits lower levels of activated Rac1 but exhibits greater Rac1 activation in response to PDGF. Serum starved and PDGF-induced Rac1 activity was evaluated by luminescence assay ; PDGF was at 100 ng/ml. Measurements were performed in triplicate, error bars represent ± s.e.m., n = 4. B. PDGF-induced Alexa Fluor 647 uptake is enhanced in Abi1 isoform expressing cell lines. Cellular accumulation of Alexa Fluor 647 in LNCaP cell lines was evaluated by flow cytometry in serum starved or PDGF (100 ng/ml) treated cells following a 2-hour incubation with the dye. Mock, mock transfected cell line; Iso-2.1, isoform 2 expressing cell line; Iso-3.1, isoform 3 expressing cell line. Measurements were performed in triplicate, n = 3, error bars ± s.e.m.

Article Snippet: Antibody 1G9 to Abi1 was from MBL International Corporation (Woburn, MA).

Techniques: Expressing, Activation Assay, Activity Assay, Luminescence Assay, Flow Cytometry, Incubation, Transfection

Journal: PLoS ONE

Article Title: Differential Regulation of Macropinocytosis by Abi1/Hssh3bp1 Isoforms

doi: 10.1371/journal.pone.0010430

Figure Lengend Snippet: A. Comparison of Rac1 mutant interactions with Abi1 isoforms. Constitutively active Rac1-V12G (GFP-Rac1-V12G) or dominant negative Rac1-T17N (GFP-Rac1-T17N), were transfected separately and analyzed in Abi1 isoform expressing LNCaP cell lines as described in . Membranes were blotted with anti-HA, anti-Sra-1, anti-Nap1 or anti-Wave2 antibodies to demonstrate the level of co-immunoprecipitated Wave 2 complex components; or with GFP antibody (GFP-Rac1-V12G, or GFP-Rac1-T17N) to demonstrate the level of immunoprecipitated Rac1. (Right panel) Immunoprecipitation results were further confirmed in affinity capture analyses where cell lysates from Iso-2.1 or Iso-3.1 expressing cell lines were subjected to pull down with either GST tagged Rac1 T17N (inactive) or Q61L (active) mutants used as baits. B. Quantification of Rac1 binding to Abi1 isoforms in immunoprecipitation experiments. Relative binding of active vs. inactive Rac1 to Abi1 isoforms was evaluated from proteins band intensities in three independent immunoprecipitation experiments. C. Alexa Fluor 647 uptake in LNCaP cells transfected with active Rac1 shows opposite effects in Iso-2.1 compared to Iso-3.1 cell lines. Measurements were performed in triplicate, n = 3, error bars ± s.e.m.

Article Snippet: Antibody 1G9 to Abi1 was from MBL International Corporation (Woburn, MA).

Techniques: Mutagenesis, Dominant Negative Mutation, Transfection, Expressing, Immunoprecipitation, Binding Assay

Journal: PLoS ONE

Article Title: Differential Regulation of Macropinocytosis by Abi1/Hssh3bp1 Isoforms

doi: 10.1371/journal.pone.0010430

Figure Lengend Snippet: A. Schematic representation of Abi1 truncated mutants used for mapping and Rac1 structure. A total of eight Abi1 constructs were used, each represents a major domain or region in the Abi1 protein . Rac1 contains three major GTP binding sites (GTPbs), two switch regions (SI and SII) and GEF/GAP/GDI binding region. Three mutations are depicted: N-terminal mutations T17N (dominant negative), V12G and Q61L (constitutively active). B. Binding of Rac1 to Abi1 truncated products. Inactive Rac1 binds to the N-terminal truncated mutant encompassing N-186 residues, and active Rac1 mutant shows exon 10 dependence in binding. Active Rac1Q61L binds weakly to this region. Active Rac1Q61L also shows exon 10 dependent binding to the 303-C region. Observations are summarized in Table below.

Article Snippet: Antibody 1G9 to Abi1 was from MBL International Corporation (Woburn, MA).

Techniques: Construct, Binding Assay, Dominant Negative Mutation, Mutagenesis

Journal: PLoS ONE

Article Title: Differential Regulation of Macropinocytosis by Abi1/Hssh3bp1 Isoforms

doi: 10.1371/journal.pone.0010430

Figure Lengend Snippet: A. Immunostaining. Cell lines ectopically expressing recombinant isoform 2 and isoform 3 were immunostained with monoclonal antibody to HA (green). Representative images from DIC (left panels) and confocal channel (middle panels) are presented; boxed areas are enlarged on the right. Nuclei were stained with TO-Pro-3 (blue). Mock, mock transfected. B. Subcellular fractionation. Distribution of Abi1 isoforms in subcellular fractions obtained by indicated centrifugation steps (1 K = 1000×g, ). Analysis by Western blotting was performed using antibodies to: Ha (Abi1); Wave2, GAPDH (GAPDH); and beta spectrin (Spectrin). Transfected cell lines Iso-2.1 and Iso-3.1 were used for isoform 2 and isoform 3 subcellular fractionation, respectively.

Article Snippet: Antibody 1G9 to Abi1 was from MBL International Corporation (Woburn, MA).

Techniques: Immunostaining, Expressing, Recombinant, Staining, Transfection, Fractionation, Centrifugation, Western Blot

Journal: PLoS ONE

Article Title: Differential Regulation of Macropinocytosis by Abi1/Hssh3bp1 Isoforms

doi: 10.1371/journal.pone.0010430

Figure Lengend Snippet: A. Abi1-Rac1 isoform-specific Wave 2 complexes. Abi1 isoforms enter Wave 2 complex in an isoform-specific manner. Both isoform 2 and isoform 3 can enter Wave 2 complex, but isoform 2 provides an additional site in exon 10 (ex10) for interaction with active Rac1 (Rac1 GTP) as suggested by in vitro binding experiments. Active Rac1 was previously demonstrated to bind to two components of Wave complex, Nap1 and Sra-1. Isoform 2 sequesters active Rac1 in cytoplasm, thus leading to lower cellular levels of active Rac1 in isoform 2 expressing cells. B. Phenotypic differences in Abi1 isoform-specific cell lines. Cell lines expressing Abi1 isoform 2 or isoform 3 cell exhibit isoform-specific phenotypes.

Article Snippet: Antibody 1G9 to Abi1 was from MBL International Corporation (Woburn, MA).

Techniques: In Vitro, Binding Assay, Expressing

Journal: Cells

Article Title: Smooth Muscle Myosin Localizes at the Leading Edge and Regulates the Redistribution of Actin-regulatory Proteins during Migration

doi: 10.3390/cells11152334

Figure Lengend Snippet: MLC 20 regulates the recruitment of c-Ab, cortactin, Pfn-1, and Abi1 to the cell edge. ( A ) Ctrl and MLC 20 KD cells were plated onto collagen-coated coverslips for 30 min followed by immunofluorescence and fluorescence analysis. MLC 20 KD attenuates the localization of c-Abl, cortactin (cort), Pfn-1, Abi1, and F-actin at the cell edge. The arrows point to the leading edge. Scale bar: 10 µm. ( B ) Illustration of quantitative analysis. An average of at least 5 line scans across each cell is used for analysis. Relative Intensity (RI) = Edge Intensity (EdI)/Cortex Intensity (CI). ( C ) Data are mean values of experiments from at least 20 cells for each group. Error bars indicate SD. One-way ANOVA was used for statistical analysis. ** p < 0.01; * p < 0.05.

Article Snippet: Abi1 antibody was purchased from Sigma (#A5106–200UL, L/N 076M4842V) and validated by using corresponding KD cells [ ].

Techniques: Immunofluorescence, Fluorescence

Journal: Cells

Article Title: Smooth Muscle Myosin Localizes at the Leading Edge and Regulates the Redistribution of Actin-regulatory Proteins during Migration

doi: 10.3390/cells11152334

Figure Lengend Snippet: MYH11 orchestrates the positioning of c-Ab, cortactin, Pfn-1, and Abi1 to the cell edge. ( A ) Ctrl and MYH11 KD cells were plated onto collagen-coated coverslips for 30 min followed by immunofluorescence and fluorescence analysis. The arrows point to the leading edge. Scale bar: 10 µm. ( B ) Data are mean values of experiments from at least 20 cells for each group. Error bars indicate SD. One-way ANOVA was used for statistical analysis. ** p < 0.01; * p < 0.05.

Article Snippet: Abi1 antibody was purchased from Sigma (#A5106–200UL, L/N 076M4842V) and validated by using corresponding KD cells [ ].

Techniques: Immunofluorescence, Fluorescence

Journal: Cells

Article Title: Smooth Muscle Myosin Localizes at the Leading Edge and Regulates the Redistribution of Actin-regulatory Proteins during Migration

doi: 10.3390/cells11152334

Figure Lengend Snippet: Proposed mechanism: In response to extracellular cues (e.g., the ECM), integrin β1 locates at the tip of lamellipodia, which recruits MLCK to the leading cell edge via an unknown mechanism. The kinase then catalyzes MLC 20 phosphorylation, which promotes the recruitment of c-Abl, cortactin, Pfn-1, and Abi1 to the leading edge, lamellipodial formation, and migration.

Article Snippet: Abi1 antibody was purchased from Sigma (#A5106–200UL, L/N 076M4842V) and validated by using corresponding KD cells [ ].

Techniques: Migration

Journal: Neoplasia (New York, N.Y.)

Article Title: The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis

doi: 10.1016/j.neo.2022.100819

Figure Lengend Snippet: Abl tyrosine kinases dependent degradation of Abi Proteins in Bcr-Abl-positive leukemic cells. A. Expression of p185 Bcr-Abl in Ba/F3 cells induces down regulation of Abi2. Total lysates from 1 × 10 6 Ba/F3 and Ba/F3 expressing p185 Bcr-Abl cells were analyzed by western blot using indicated antibodies. B. Abl tyrosine kinase inhibitor imatinib (IM) reverts Bcr-Abl-induced down-regulation of Abi and WAVE2 proteins. Ba/F3 p185 Bcr-Abl and K562 cells were treated with or without 5 μM Abl kinase inhibitor imatinib (IM) for 8 h. Total lysates of 1 × 10 6 cells were analyzed by western blot using indicated antibodies. C. Imatinib (IM) treatment increases Abi1 protein level in p185 Bcr-Abl -positive leukemic cells. The p185 Bcr-Abl cells expressing HA-tagged Abi1 were treated with or without 5 μM Abl kinase inhibitor imatinib (IM), as indicated, at the presence of 50 μM cycloheximide (CHX) for indicated hours. Total lysates of 1 × 10 6 cells were analyzed by western blot using indicated antibodies. D. Proteasome inhibitors revert Bcr-Abl-induced Abi2 down regulation. The p185 Bcr-Abl cells were treated with proteasome inhibitors MG132 (20 μM) and lactacystin (10 μM), lysosome inhibitors bafilomycin A1 (1 μM) and chloroquine (100> μM), and calpain inhibitor ALLN (25 μM), as indicated, for 5 >h. Total lysates from 1 × 10 6 cells were subjected to western blot analysis.

Article Snippet: The rabbit polyclonal antibodies against Abi1 phospho-tyrosine 213 was generated with Proteintech Group Inc. (Chicago, IL) using the tyrosine 213 phospho-peptide VKPPTVPNDY(PO 4 ) MTSPARLG as antigen and was purified by affinity chromatography column.

Techniques: Expressing, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis

doi: 10.1016/j.neo.2022.100819

Figure Lengend Snippet: The phosphorylation of tyrosine 213 is required for Bcr-Abl-induced degradation of Abi1 and Abi2. A. Phosphorylation of Abi1 tyrosine 213 (Y213) in p185 Bcr-Abl cells. Ba/F3 cells and Ba/F3 cells expressing p185 Bcr-Abl or p185 Bcr-Abl plus green fluorescence protein (GFP)-tagged Abi1, as indicated, were treated with or without 5 μM imatinib (IM) for 8 h. Cell lysates were subjected to immunoprecipitation (IP) using anti-Abi1 antibody and the immunoprecipitates were analyzed by western blotting using an antibody raised against a peptide flanking phosphorylated tyrosine 213 (pY213). B. A mutation of Y213 to phenylalanine (Y213F) increases Abi1 protein stability in Bcr-Abl-positive leukemic cells. The p185 Bcr-Abl cells expressing the HA-tagged wild type Abi1 (p185 HA-Abi1wt, upper panel) or Y213F mutant (p185 HA-Abi1Y213F, lower panel) were treated with 50 μM cycloheximide for indicated hours. Total lysates of 1 × 10 6 cells were analyzed by western blot using indicated antibodies. C. A mutation of Y213 to phenylalanine (Y213F) abolishes Bcr-Abl-induced degradation of Abi2. The p185 Bcr-Abl cells stably expressing the HA-tagged wild type Abi2 (HA-Abi2 wt) or Y213F mutant (HA-Abi2Y213F) were treated with or without 5 μM imatinib (IM) for 8 h. Total lysates of 1 × 10 6 cells were analyzed by western blot using indicated antibodies. D. The Y213F mutation reduces Abi2 ubiquitination in p185 Bcr-Abl cells. The p185 Bcr-Abl cells stably expressing the HA-tagged wild type Abi2 (HA-Abi2 wt) or Y213F mutant (HA-Abi2Y213F) were treated with or without 50 μM MG132 for 5 h. Cell lysates were subjected to immunoprecipitation (IP) using anti-HA antibody and the immunoprecipitates were analyzed by western blotting using the indicated antibodies.

Article Snippet: The rabbit polyclonal antibodies against Abi1 phospho-tyrosine 213 was generated with Proteintech Group Inc. (Chicago, IL) using the tyrosine 213 phospho-peptide VKPPTVPNDY(PO 4 ) MTSPARLG as antigen and was purified by affinity chromatography column.

Techniques: Phospho-proteomics, Expressing, Fluorescence, Immunoprecipitation, Western Blot, Mutagenesis, Stable Transfection, Ubiquitin Proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis

doi: 10.1016/j.neo.2022.100819

Figure Lengend Snippet: Abi proteins contain a Cbl-TKB binding motif and Bcr-Abl induced downregulation of WRC requires Cbl E3 ubiquitin ligase. A. The alignment of amino acid sequences found in Abi1, Abi2, and various proteins that have been shown to bind to the Cbl-TKB domain. The consensus motif found in a cohort of protein tyrosine kinases (PTK) family is shown at the bottom. The highly conserved aspartic acid (D)/asparagine (N), tyrosine (Y), and proline (P) are in green, red, and purple, respectively, whereas the X represents any amino acid. B. Double knockout of c-Cbl and Cbl-B in p185 Bcr-Abl cells rescues Abi2 from Bcr-Abl-induced degradation. Total lysates from 1 × 10 6 Ba/F3, p185 Bcr-Abl control (p185 ctrl), and two independent lines of p185 Bcr-Abl cells in which c-Cbl (Cbl KO1 and KO2), Cbl-B (Cbl-B KO1 and KO2), or both c-Cbl and Cbl-B (Cbl/Cbl-B KO1 and KO2) have been knocked out by CRISPR/Cas9-mediated gene editing were subjected to western blot analysis using the indicated antibodies. C. Double knockout of c-Cbl and Cbl-B in p185 Bcr-Abl cells increases protein levels of Abi1, Abi2, and WAVE2. Total lysates from 1 × 10 6 p185 Bcr-Abl control (p185 ctrl) and two independent lines of p185 Bcr-Abl c-Cbl/Cbl-B double knockout cells (Cbl/Cbl-B KO1 and KO2) were subjected to western blot analysis using the indicated antibodies. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The rabbit polyclonal antibodies against Abi1 phospho-tyrosine 213 was generated with Proteintech Group Inc. (Chicago, IL) using the tyrosine 213 phospho-peptide VKPPTVPNDY(PO 4 ) MTSPARLG as antigen and was purified by affinity chromatography column.

Techniques: Binding Assay, Ubiquitin Proteomics, Double Knockout, Control, CRISPR, Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis

doi: 10.1016/j.neo.2022.100819

Figure Lengend Snippet: Abi1 depletion downregulates WAVE2 and inhibits Akt and Lyn pathways in Bcr-Abl positive leukemic cells. A. Downregulation of WAVE2 in Abi1-depleted K562 cells. Total lysates from 1 × 10 6 control K562 (K562 ctrl) and three independent clones of Abi1-depleted K562 cells (K562 KO B4, E4, and E3) were analyzed by western blot using indicated antibodies. B. Depletion of Abi1 in p185 Bcr-Abl cells (left panel) and K562 cells (right panel) reduces Lyn activation. Total lysates from 1 × 10 6 p185 Bcr-Abl control cells (p185 ctrl), K562 control cells (K562 ctrl), two independent clones of Abi1-depleted p185 Bcr-Abl cells (Abi1 KO2.3 and Abi1 KO6.2), and two independent clones of Abi1-depleted K562 (K562 KO B4 and KO E4) were analyzed by western blot using indicated antibodies. C. Abi1 knockout in K562 cells inhibits the activation of Akt pathway. Total lysates from 1 × 10 6 K562 control (K562 ctrl) and three clones of Abi1-depleted K562 cells (K562 KO B4, KO E4 and KO E3) were analyzed by western blot using indicated antibodies.

Article Snippet: The rabbit polyclonal antibodies against Abi1 phospho-tyrosine 213 was generated with Proteintech Group Inc. (Chicago, IL) using the tyrosine 213 phospho-peptide VKPPTVPNDY(PO 4 ) MTSPARLG as antigen and was purified by affinity chromatography column.

Techniques: Control, Clone Assay, Western Blot, Activation Assay, Knock-Out

Journal: Neoplasia (New York, N.Y.)

Article Title: The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis

doi: 10.1016/j.neo.2022.100819

Figure Lengend Snippet: Abi1 is involved in the regulation of the EGF/EGFR signaling in metastatic breast cancer cells. A. Abi1 binds to EGFR in metastatic MDA-MB-231 human breast cancer cells upon EGF stimulation. A brain metastatic subline of MDA-MB-231 cells (231Br) was stimulated with or without 50 ng/ml EGF for 30 min. The total lysates were subjected to immunoprecipitation (IP) followed by western blot (WB) analysis using antibodies as indicated. B. Abi1 is tyrosine-phosphorylated and the Y213 is a major site phosphorylated by Abl tyrosine kinases in metastatic breast cancer cells. Left panel: lysates of human breast cancer cell lines MCF-7 (MCF-7), MDA-MB-231 (MB231), and a brain metastatic subline of MDA-MB-231 (231Br) cells were subjected to immunoprecipitation (IP) followed by western blot (WB) analysis using indicated antibodies. Right panel: A lung metastatic subline of MDA-MB-231 cells, LM2-4175 was transduced with retroviruses expressing GFP-tagged wild type Abi1 (WT) or Abi1 Y213F mutant (Y213F). The cells were treated with or without 5 μM imatinib (IM), as indicated, for 8 h and total cell lysates were subjected to immunoprecipitation (IP) followed by western blot (WB) analysis using indicated antibodies. C and D. CRISPR/Cas9 mediated knockout of Abi1 in LM2-4175 breast cancer cells leads to EGFR down regulation. The control LM2-4175 cells (475 ctrl) and Abi1-depleted LM2-4175 cells (4175 KO) were stimulated with 50 ng/ml EGF for 60 min (C) or indicated time (D). Total cell lysates were analyzed by western blotting with indicated antibodies.

Article Snippet: The rabbit polyclonal antibodies against Abi1 phospho-tyrosine 213 was generated with Proteintech Group Inc. (Chicago, IL) using the tyrosine 213 phospho-peptide VKPPTVPNDY(PO 4 ) MTSPARLG as antigen and was purified by affinity chromatography column.

Techniques: Immunoprecipitation, Western Blot, Transduction, Expressing, Mutagenesis, CRISPR, Knock-Out, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis

doi: 10.1016/j.neo.2022.100819

Figure Lengend Snippet: Abi1 depletion impairs LM2-4175 breast cancer cells invasion in vitro. A. A 3D tumor spheroid invasion assay of control LM2-4175 cells (4175 Control) and 2 individual clones of LM2-4175 Abi1 knockout cells (4175 Abi1 KO1 and 4175 Abi1 KO2). The data is a representative of three independent experiments. B. Comparison of invasive area of control LM2-4175 cells (4175 Ctrl) and LM2-4175 Abi1 knockout cells (4175 Abi1 KO1 and 4175 Abi1 KO2) in a triplicate experiment. The invasion area was calculated using Adobe Photoshop software. Unpaired student t test was used to compare the knockout cells with the 4175 controls. * P = 0.18 ; ** P < 0.05 ; *** P < 0.01

Article Snippet: The rabbit polyclonal antibodies against Abi1 phospho-tyrosine 213 was generated with Proteintech Group Inc. (Chicago, IL) using the tyrosine 213 phospho-peptide VKPPTVPNDY(PO 4 ) MTSPARLG as antigen and was purified by affinity chromatography column.

Techniques: In Vitro, Invasion Assay, Control, Clone Assay, Knock-Out, Comparison, Software

Journal: Neoplasia (New York, N.Y.)

Article Title: The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis

doi: 10.1016/j.neo.2022.100819

Figure Lengend Snippet: Abi1 knockout impedes LM2-4175 breast cancer cells metastasis to lung in vivo. A. Lung weight of the mice injected intracardiacally with control LM2-4175 cells (4175 Ctrl, n = 5) and 2 individual clones of LM2-4175 Abi1 knockout cells (4175 KO1, n = 5; and 4175 KO2, n = 5; * P <0.05 compared to 4175 Ctrl). B. H&E staining of the representative lungs from the mouse injected with saline (control), LM2-4175 control cells (4175 Ctrl), and two clones of LM2-4175 Abi1 knockout cells (4175 KO1 and 4175 KO2). C. Bioluminescence imaging of mice at 2, 7, 10, 14, 17, and 20 days after intracardiac injection of LM2-4175 (LM2-4175 Ctrl) or LM2-4175 Abi1 knockout (LM2-4175 Abi1 KO) cells. Scale bar denotes radiance in photons per second per square centimeter per steradian (p/sec/cm 2 /sr). D. The quantitative bioluminescence imaging as the value of total flux (photons/sec) for mice injected with LM2-4175 control (red) and Abi1 knockout (4175 Abi1 KO, blue) cells. E. Survival curve of the mice injected with LM2-4175 cells (red, n = 5) and two individual clones of LM2-4175 Abi1 knockout cells, 4175 KO1 and 4175 KO2 (blue and purple, respectively, n = 5 for each group). Log-rank test P < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The rabbit polyclonal antibodies against Abi1 phospho-tyrosine 213 was generated with Proteintech Group Inc. (Chicago, IL) using the tyrosine 213 phospho-peptide VKPPTVPNDY(PO 4 ) MTSPARLG as antigen and was purified by affinity chromatography column.

Techniques: Knock-Out, In Vivo, Injection, Control, Clone Assay, Staining, Saline, Imaging

Journal: Neoplasia (New York, N.Y.)

Article Title: The Abl/Abi signaling links WAVE regulatory complex to Cbl E3 ubiquitin ligase and is essential for breast cancer cell metastasis

doi: 10.1016/j.neo.2022.100819

Figure Lengend Snippet: Summary of the disease development in mice injected with LM2-4175 control cells and the LM2-4175 Abi1 KO cells.

Article Snippet: The rabbit polyclonal antibodies against Abi1 phospho-tyrosine 213 was generated with Proteintech Group Inc. (Chicago, IL) using the tyrosine 213 phospho-peptide VKPPTVPNDY(PO 4 ) MTSPARLG as antigen and was purified by affinity chromatography column.

Techniques: Injection, Control, Saline