Journal: Cancer Cell International

Article Title: Inhibition of ABI2 ubiquitination-dependent degradation suppresses TNBC cell growth via down-regulating PI3K/Akt signaling pathway

doi: 10.1186/s12935-024-03407-0

Figure Lengend Snippet: The expression of ABI2 is markedly reduced in patients with TNBC. ( a ) Heatmap analysis of the 279 ubiquitinated human substrate proteins expression in Basal subtype of breast cancer; ( b ) Venn diagram analysis of differentially-expressed substrate proteins in Basal, LumA, LumB and Her2 subtypes of breast cancer; ( c ) The 14 distinctively-expressed proteins in the Basal subtype of breast cancer; ( d ) Kaplan- Meier curve of ABI2 in the survival analysis of patients with TNBC. ( e ) IHC analysis of ABI2 protein expression in TNBC tumors. ( f ) Western blotting analysis of ABI2 protein expression in MDA-MB-231, MCF-10 A and MCF-7 cells

Article Snippet: ABI2 immunohistochemistry (IHC) staining was conducted using established protocols, with a ABI2 antibody diluted at 1:100 (Santa Cruz, No. sc-393,982; Dallas, USA).

Techniques: Expressing, Western Blot

Journal: Cancer Cell International

Article Title: Inhibition of ABI2 ubiquitination-dependent degradation suppresses TNBC cell growth via down-regulating PI3K/Akt signaling pathway

doi: 10.1186/s12935-024-03407-0

Figure Lengend Snippet: AB12 inhibits cell viability, migration, invasion and arrests cell cycle of TNBC cells. ( a ) Effects of OE-ABI2 on the growth pattern of MDA-MB-231 cells detected by CCK-8 method; ( b ) Effects of OE-ABI2 on the cell migration of MDA-MB-231 cells detected by wound-healing assay; ( c ) Effects of OE-ABI2 on the cell invasion of MDA-MB-231 cells detected by Transwell assay; ( d ) Effects of OE-ABI2 on the cell cycle of MDA-MB-231 cells detected by flow cytometry. Data are presented as the mean ± SD. ** p < 0.01 vs. NC group

Article Snippet: ABI2 immunohistochemistry (IHC) staining was conducted using established protocols, with a ABI2 antibody diluted at 1:100 (Santa Cruz, No. sc-393,982; Dallas, USA).

Techniques: Migration, CCK-8 Assay, Wound Healing Assay, Transwell Assay, Flow Cytometry

Journal: Cancer Cell International

Article Title: Inhibition of ABI2 ubiquitination-dependent degradation suppresses TNBC cell growth via down-regulating PI3K/Akt signaling pathway

doi: 10.1186/s12935-024-03407-0

Figure Lengend Snippet: ABI2 inhibits PI3K/Akt signaling pathway in TNBC cells. ( a ) Flowchart of RNA-seq analysis on MDA-MB-231 cells treated with OE-ABI2; ( b ) Volcano diagram analysis of differentially expressed genes in MDA-MB-231 cells with OE-ABI2; ( c ) KEGG analysis of differentially expressed genes in MDA-MB-231 cells with OE-ABI2; ( d ) Heatmap analysis of differentially expressed genes of PI3K/Akt and Hippo signaling pathways in MDA-MB-231 cells with OE-ABI2; ( e ) qPCR validation of differentially expressed genes of PI3K/Akt and Hippo signaling pathways in MDA-MB-231 cells with OE-ABI2. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01 vs. NC group

Article Snippet: ABI2 immunohistochemistry (IHC) staining was conducted using established protocols, with a ABI2 antibody diluted at 1:100 (Santa Cruz, No. sc-393,982; Dallas, USA).

Techniques: RNA Sequencing, Protein-Protein interactions, Biomarker Discovery

Journal: Cancer Cell International

Article Title: Inhibition of ABI2 ubiquitination-dependent degradation suppresses TNBC cell growth via down-regulating PI3K/Akt signaling pathway

doi: 10.1186/s12935-024-03407-0

Figure Lengend Snippet: ABI2 may regulate PI3K/Akt signaling pathway through interacting with RAC1 protein in TNBC cells. ( a ) ABI2 protein expression validation in MDA-MB-231 cells; ( b ) ABI2 protein expression after IP experiment; ( c ) The silver staining of proteins in IP experimental group and IgG control group; ( d ) Venn diagram of differential proteins between IP experimental group and IgG control group; ( e ) KEGG analysis of signaling pathways involved in the differential proteins; ( f ) Co-IP results of the interaction between ABI2 and RAC1 proteins

Article Snippet: ABI2 immunohistochemistry (IHC) staining was conducted using established protocols, with a ABI2 antibody diluted at 1:100 (Santa Cruz, No. sc-393,982; Dallas, USA).

Techniques: Expressing, Biomarker Discovery, Silver Staining, Control, Protein-Protein interactions, Co-Immunoprecipitation Assay

Journal: Cancer Cell International

Article Title: Inhibition of ABI2 ubiquitination-dependent degradation suppresses TNBC cell growth via down-regulating PI3K/Akt signaling pathway

doi: 10.1186/s12935-024-03407-0

Figure Lengend Snippet: Up-regulation of ABI2 via inhibiting CBLC mediated-ubiquitination may be potential treatment for TNBC. ( a ) CBLC was predicted to be the E3 ligases of ABI2 protein by using bioinformatics; ( b ) There was a significant co-precipitation between the natural forms of ABI2 and CBLC in HEK293T cells through the employment of the Co-IP technique; ( c ) The exogenous and endogenous ubiquitination experiments with or without MG132. ( d-e ) CBLC could promote the binding of ABI2 to Ub and increase the ubiquitization degradation of ABI2 protein at both exogenous and endogenous levels of ubiquitination; ( f ) Molecular docking mode and amino acid interaction residues of ABI2 and CBLC proteins; ( g ) Virtual screening process from 2,800 FDA-approved drug molecules based on the molecular docking mode of ABI2 and CBLC proteins; ( h ) Top 9 molecules with the best ranking between ABI2 and CBLC proteins

Article Snippet: ABI2 immunohistochemistry (IHC) staining was conducted using established protocols, with a ABI2 antibody diluted at 1:100 (Santa Cruz, No. sc-393,982; Dallas, USA).

Techniques: Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Binding Assay

Journal: Cancer Cell International

Article Title: Inhibition of ABI2 ubiquitination-dependent degradation suppresses TNBC cell growth via down-regulating PI3K/Akt signaling pathway

doi: 10.1186/s12935-024-03407-0

Figure Lengend Snippet: CS suppresses TNBC cell growth through inhibiting ABI2-CBLC protein interaction. ( a-b ) Effects of top molecules including Atosiban Acetate, Bleomycin Sulfate, Degarelix Acetate, Ganirelix Acetate and CS on MDA-MB-231 cells detected by CCK-8 method; ( c ) Effects of CS on the growth pattern of MDA-MB-231 cells detected by CCK-8 method; ( d ) Effects of CS on the cell migration of MDA-MB-231 cells detected by wound-healing assay; ( e ) Effects of CS on the cell cycle of MDA-MB-231 cells detected by flow cytometry; ( f ) Molecular docking mode and amino acid interaction residues of ABI2 protein and CS. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01 vs. Ctrl group

Article Snippet: ABI2 immunohistochemistry (IHC) staining was conducted using established protocols, with a ABI2 antibody diluted at 1:100 (Santa Cruz, No. sc-393,982; Dallas, USA).

Techniques: CCK-8 Assay, Migration, Wound Healing Assay, Flow Cytometry

Journal: Cancer Cell International

Article Title: Inhibition of ABI2 ubiquitination-dependent degradation suppresses TNBC cell growth via down-regulating PI3K/Akt signaling pathway

doi: 10.1186/s12935-024-03407-0

Figure Lengend Snippet: CS induces TNBC cells apoptosis via inhibition of PI3K/Akt signaling pathway. ( a-b ) Effects of CS and OE-ABI2 on PI3K/Akt signaling pathway of MDA-MB-231 cells detected by western blotting assay; ( c-d ) Effects of CS and OE-ABI2 on the apoptosis of MDA-MB-231 cells detected by TUNEL method; ( e ) Effects of CS and OE-ABI2 on the cell migration of MDA-MB-231 cells detected by wound-healing assay; ( f ) Effects of CS and OE-ABI2 on the apoptosis of MDA-MB-231 cells detected by flow cytometry. Data are presented as the mean ± SD. * p < 0.05, ** p < 0.01 vs. Ctrl group

Article Snippet: ABI2 immunohistochemistry (IHC) staining was conducted using established protocols, with a ABI2 antibody diluted at 1:100 (Santa Cruz, No. sc-393,982; Dallas, USA).

Techniques: Inhibition, Western Blot, TUNEL Assay, Migration, Wound Healing Assay, Flow Cytometry

Journal: Cancer Cell International

Article Title: Inhibition of ABI2 ubiquitination-dependent degradation suppresses TNBC cell growth via down-regulating PI3K/Akt signaling pathway

doi: 10.1186/s12935-024-03407-0

Figure Lengend Snippet: The molecular mechanism diagram of this study. ABI2, acting as a crucial factor of tumor suppression, hinders the initiation and progression of TNBC through inhibiting PI3K/Akt pathway via the interaction with RAC1 protein. Furthermore, the FDA-approved drug CS has shown significant potential in suppressing the proliferation of TNBC cells and inducing cell apoptosis by inhibiting the ubiquitination modification of ABI2 protein mediated by CBLC E3 ligase

Article Snippet: ABI2 immunohistochemistry (IHC) staining was conducted using established protocols, with a ABI2 antibody diluted at 1:100 (Santa Cruz, No. sc-393,982; Dallas, USA).

Techniques: Ubiquitin Proteomics, Modification

Journal: bioRxiv

Article Title: GTP signaling links metabolism, DNA repair, and responses to genotoxic stress

doi: 10.1101/2023.04.12.536297

Figure Lengend Snippet: (A) Schematic of phosphoproteomic assay, analysis pipeline and nomination of Abi-1-S323. (B) Levels of p-Abi-1-S323 as determined by phosphoproteomic assay. There is a DNA damage-induced dephosphorylation of Abi-1-S323 that is augmented by GTP supplementation but blocked by GTP deprivation with mycophenolic acid (MPA). The effects of MPA are abrogated when GTP supplementation is combined with MPA treatment. (C) Cells were treated with MPA (10 μm) and/or G (50 μm) for 24 h and retreated with G (50 μm) 2 h before RT, followed by cell harvesting 4 h after RT for immunoblot assay. (D&E) Control knockout (Cont-KO) or Abi-1 knockout (Abi-1-KO) cells were treated with MPA, G, and/or RT as before and harvested to assess γ-H2AX levels by immunoblot (D) or foci quantification (E). (F&G) Abi-1-KO cells were transfected with plasmids encoding wild type Abi-1, phospho-mimetic Abi-1 (S323D) or dephospho-mimetic Abi-1 (S323A) and treated as above, followed by immunoblot (F) or γ-H2AX foci assay (G). (H&I) Abi-1-KO cells were transfected with individual Abi-1 plasmid and treated with MPA and/or G overnight and retreated with G 2 h before transfection of linearized pEYFP products, followed by cell harvesting 24 h post transfection and qPCR assay with data normalized to untreated control cells. Data are presented as mean ± SEM from three biologically independent experiments for Figure E, G, H, I, and Figure C, D, F are representative figures from three biologically independent experiments. Two-tailed unpaired student’s t test *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: SiRNAs of pan PP1 (a pool of 6 siRNAs, sc-43545), PP2A-Cα (a pool of 3 siRNAs, sc-43509), PP2A-Cβ (a pool of 3 siRNAs, sc-36301), PP4 (a pool of 3 siRNAs, sc-39202), PP5 (a pool of 3 siRNAs, sc-44602), Abi-1 (sc-417534-NIC) double nickase CRISPR/CAS9 knockout plasmids and control (sc-437281) were obtained from Santa Cruz Biotechnology.

Techniques: De-Phosphorylation Assay, Cell Harvesting, Western Blot, Control, Knock-Out, Transfection, Plasmid Preparation, Two Tailed Test

Journal: bioRxiv

Article Title: GTP signaling links metabolism, DNA repair, and responses to genotoxic stress

doi: 10.1101/2023.04.12.536297

Figure Lengend Snippet: (A-F) IHC staining was performed to detect expression of p-Abi-1 and two downstream proteins of active Rac1 (PAK1 and PAK2) in GBM PDX tissue arrays. Survival FC indicates the fold increase in median survival with the indicated treatment compared to placebo control. Each dot indicates a different murine PDX model. (G) A schematic timeline of GBM38 orthotopic mouse models (additional details in Methods). (H-K) A subset of mice (3-4 mice/group) were treated with three doses of the Rac1 inhibitor MBQ-167 and two fractions (2 Gy/fraction) of radiation and tumors were harvested 4 h after receiving the second RT dose for IHC staining with indicated antibodies. (L-N) Another subset mice (7-10 mice/group) were treated with 12 doses of MBQ-167 and 10 fractions (2 Gy/fraction) of radiation (as shown in ), were used for efficacy evaluation. Mice were treated with 150 mg/kg D-luciferin and imaged 10 min post-injection (L). Total flux of equal-area ROIs at each time point were normalized to flux at the first day of treatment for evaluating tumor progression (M). Mice were monitored daily and euthanized when they developed neurologic symptoms and Kaplan–Meier survival curve was plotted (N). Data are presented as mean ± SEM from 3-4 independent mice for Figure H-K and 7-10 mice for figure L-N. Two-tailed unpaired student’s t test **p < 0.01, ***p < 0.001, ****p < 0.0001 for and H-K; Log-rank (Mantel-Cox) test *p < 0.05, **p < 0.01, ***p < 0.001 for .

Article Snippet: SiRNAs of pan PP1 (a pool of 6 siRNAs, sc-43545), PP2A-Cα (a pool of 3 siRNAs, sc-43509), PP2A-Cβ (a pool of 3 siRNAs, sc-36301), PP4 (a pool of 3 siRNAs, sc-39202), PP5 (a pool of 3 siRNAs, sc-44602), Abi-1 (sc-417534-NIC) double nickase CRISPR/CAS9 knockout plasmids and control (sc-437281) were obtained from Santa Cruz Biotechnology.

Techniques: Immunohistochemistry, Expressing, Control, Injection, Two Tailed Test

Journal: bioRxiv

Article Title: GTP signaling links metabolism, DNA repair, and responses to genotoxic stress

doi: 10.1101/2023.04.12.536297

Figure Lengend Snippet: (A-D) Con-KO or Abi-1-KO cells were treated with different doses of radiation (A) or escalating concentrations of bleomycin (B; Con-KO vs Abi-KO IC50:0.77 ± 0.07 vs 0.30 ± 0.09, p = 0.02), TMZ alone (C; 265.33 ± 34.77 vs 134.60 ± 25.71, p = 0.04) or TMZ combined with RT (4 Gy) (D; 229.23 ± 41.65 vs 75.68 ± 21.61, p = 0.03) and cell viability was evaluated with long-term cell viability assay at day 7 post treatment. All drug concentrations are in μM. (E-H) Abi-KO cells were transiently transfected with Abi-1-WT, -S323D or -S323A, followed by irradiation (E), bleomycin (F; IC50 of WT vs S323D:1.13 ± 0.11 vs 0.28 ± 0.004, p = 0.03; WT vs S323A:1.13 ± 0.11 vs 4.32 ± 0.33, p = 0.02), TMZ alone (G; IC50 of WT vs S323D: 324.53 ± 16.43 vs 90.16 ± 38.91, p = 0.005; WT vs S323A: 324.53 ± 16.43 vs 425.73 ± 23.61, p = 0.02) or TMZ combined with RT (4 Gy) (H; IC50 of WT vs S323D:215.13 ± 46.12 vs 47.19 ± 24.61, p=0.03; WT vs S323A: 215.13 ± 46.12 vs 392.43 ± 44.64, p = 0.05) treatment as discussed above, all drug concentrations in μM. (I-K) Luciferase-positive, cont or Abi-1 knockout, and RT-resistant GBM38 patient-derived xenograft cells were orthotopically implanted and tumor-bearing mice were randomized (7-10 animals per group). RT (2 Gy/fraction and 6 fractions for total) was given. Tumors were imaged after D-luciferin injection and mouse survival was monitored (mean ± SEM). Figure A to H are representative figures from two to three biologically independent experiments (mean ± SEM) and I-K are presented as mean ± SEM from 7-10 independent mice. Two-tailed unpaired student’s t test *p < 0.05, **p < 0.01, ***p < 0.001, for ; Log-rank (Mantel-Cox) test *p < 0.05, for .

Article Snippet: SiRNAs of pan PP1 (a pool of 6 siRNAs, sc-43545), PP2A-Cα (a pool of 3 siRNAs, sc-43509), PP2A-Cβ (a pool of 3 siRNAs, sc-36301), PP4 (a pool of 3 siRNAs, sc-39202), PP5 (a pool of 3 siRNAs, sc-44602), Abi-1 (sc-417534-NIC) double nickase CRISPR/CAS9 knockout plasmids and control (sc-437281) were obtained from Santa Cruz Biotechnology.

Techniques: Viability Assay, Transfection, Irradiation, Luciferase, Knock-Out, Derivative Assay, Injection, Two Tailed Test

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: Cell Communication and Signaling

Article Title: C3G forms complexes with Bcr-Abl and p38α MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion

doi: 10.1186/1478-811x-11-9

Figure Lengend Snippet: Figure 1 Bcr-Abl and C3G interact with Cbl, Abi1 and p130Cas. (A) Whole cell extracts from K562 were subjected to pull-down assays using GST-Abl-SH3 construct. The presence of C3G, Abi1, Cbl and p130Cas in the complexes was detected by inmunoblotting with specific antibodies. Representative Western blots of pull-down assays in K562 lysates using (B) Abi1-SH3 and SH3-b domains, (C) p130Cas-SH3, P1 and P2 domains, and (D) Cbl-SH3-b domain fused to GST as baits. The immunoblots were developed with specific antibodies against the indicated proteins. Glutathione-sepharose beads with whole cell lysate (B + K562), lysis buffer with the corresponding GST-fused proteins (LB) and pGEX-4T-1 empty plasmid with K562 whole cell lysate (GST) were used as negative controls. L: whole cell lysate (40 μg). The asterisks indicate unspecific bands.

Article Snippet: Expression of C3G, Abi1 and Cbl was silenced in K562 cells by transfection of (h) Lentiviral Particles: C3G shRNA (sc-29863-V), Abi1 shRNA (sc-40306-V), Cbl shRNA (sc-29242-V) and control shRNA (sc-108080) from Santa Cruz Biotechnology, following the manufacturer`s protocol.

Techniques: Construct, Western Blot, Lysis, Plasmid Preparation

Journal: Cell Communication and Signaling

Article Title: C3G forms complexes with Bcr-Abl and p38α MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion

doi: 10.1186/1478-811x-11-9

Figure Lengend Snippet: Figure 2 C3G colocalizes with phospho-p130Cas, Cbl, Abi1 and Bcr-Abl in K562 cells. Confocal microscopy images of K562 cells adhered to fibronectin covered slides (10 μg/ml) and labeled with anti-C3G (G4, mouse monoclonal, rows 1 and 2 or 1008, rabbit polyclonal, rows 3 and 4) and either, anti-phospho-p130Cas (rabbit polyclonal), anti-Cbl (rabbit polyclonal), anti-Abi1 (mouse monoclonal) or anti-Abl (mouse monoclonal) specific antibodies as indicated. Anti-FITC and anti-Cy3 were used as secondary antibodies. Nuclei were labeled with DAPI. Control cells (CT) were incubated with DAPI plus the secondary antibodies. DIC: Differential interference contrast microscopy. p-p130Cas: phospho-p130Cas. The bars represent 10 μm.

Article Snippet: Expression of C3G, Abi1 and Cbl was silenced in K562 cells by transfection of (h) Lentiviral Particles: C3G shRNA (sc-29863-V), Abi1 shRNA (sc-40306-V), Cbl shRNA (sc-29242-V) and control shRNA (sc-108080) from Santa Cruz Biotechnology, following the manufacturer`s protocol.

Techniques: Confocal Microscopy, Labeling, Control, Incubation, Microscopy

Journal: Cell Communication and Signaling

Article Title: C3G forms complexes with Bcr-Abl and p38α MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion

doi: 10.1186/1478-811x-11-9

Figure Lengend Snippet: Figure 3 Abi1 and p130Cas silencing alter Bcr-Abl/C3G interaction. (A) Western blot analysis of the expression of Abi1, Cbl, p130Cas and p87C3G in whole cell lysates from K562 clones stably transfected with the indicated lentiviral shRNA particles. Relative Abi1/β-tubulin, Cbl/β- tubulin, p130Cas/β-tubulin and C3G/β-tubulin ratios are shown. shAbi1-2, shCbl-1, shp130Cas-5, shC3G-1 and shC3G-8 were selected as representative knock-down clones in the experiments. C: whole cell lysate of a K562 clone expressing control lentiviral shRNA. Pull-down assays with lysates of K562 shAbi1-2, shCbl-1 and shControl (shCT) clones (B) or K562 shp130Cas-5 and shCT clones (C), using Abl-SH3 domain fused to GST as bait. p140C3G, p87C3G, Abi1, CrkL, Cbl and p130Cas expression was detected with specific antibodies. Panels showing p140C3G and p87C3G in Figure 3B correspond to different exposure times (longer for p140 and shorter for p87) for a better visualization of both isoforms. Beads with lysis buffer (B), GST construct with K562 whole cell lysate (GST) and K562 whole cell lysate with lysis buffer (LB) were used as negative controls. L: whole cell lysate (40 μg).

Article Snippet: Expression of C3G, Abi1 and Cbl was silenced in K562 cells by transfection of (h) Lentiviral Particles: C3G shRNA (sc-29863-V), Abi1 shRNA (sc-40306-V), Cbl shRNA (sc-29242-V) and control shRNA (sc-108080) from Santa Cruz Biotechnology, following the manufacturer`s protocol.

Techniques: Western Blot, Expressing, Clone Assay, Stable Transfection, Transfection, shRNA, Knockdown, Control, Lysis, Construct

Journal: Cell Communication and Signaling

Article Title: C3G forms complexes with Bcr-Abl and p38α MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion

doi: 10.1186/1478-811x-11-9

Figure Lengend Snippet: Figure 4 C3G, Cbl, Abi1, p130Cas and p38α MAPK regulate adhesion to fibronectin in K562 cells. (A) Percentage of adhesion to fibronectin of K562 cells upon knock-down of C3G (shC3G-1 and shC3G-8), Cbl (shCbl-1), Abi1 (Abi1-2) or p130Cas (shp130Cas-5). (B) Percentage of adhesion to fibronectin of K562 clones expressing interference RNAs (cloned in pSuper.gfp/neo) for C3G and/or p38α MAPK. The values are the mean ± SEM of at least three independent experiments. All values are relative to K562 cells transfected with control shRNA or empty pSuper.gfp/ neo vector (pSuper). *p<0.05; ** p<0,01.

Article Snippet: Expression of C3G, Abi1 and Cbl was silenced in K562 cells by transfection of (h) Lentiviral Particles: C3G shRNA (sc-29863-V), Abi1 shRNA (sc-40306-V), Cbl shRNA (sc-29242-V) and control shRNA (sc-108080) from Santa Cruz Biotechnology, following the manufacturer`s protocol.

Techniques: Knockdown, Clone Assay, Expressing, Transfection, Control, shRNA, Plasmid Preparation

Journal: Cell Communication and Signaling

Article Title: C3G forms complexes with Bcr-Abl and p38α MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion

doi: 10.1186/1478-811x-11-9

Figure Lengend Snippet: Figure 5 C3G and p38α MAPK interact with FA proteins. (A) Representative immunoprecipitation assays (IP), using the indicated antibodies, performed with protein extracts from K562 cells cultured on 10 μg/ml fibronectin for 24 h. Expression of p33, p46 and p68 phospho-paxillin (p-Pax) and paxillin isoforms, p38α, p87C3G, CrkL, Bcr-Abl, p130Cas, Abi1 and FAK was detected by immunoblotting with specific antibodies. GammaBind G Sepharose beads (B) with either buffer or whole cell lysate (lysate) were used as negative controls. (B) Confocal microscopy of K562 cells adhered for 24 h to slides covered with 10 μg/ml fibronectin and labeled with the indicated antibodies. Nuclei were labeled with DAPI. Control cells (CT) were incubated with DAPI plus the secondary antibodies. C3G-1008 (rabbit polyclonal) was used with Pax (mouse monoclonal) and p-FAK (goat polyclonal). C3G-G4 (mouse monoclonal) was used with p-Pax (rabbit polyclonal). C3G colocalizes with p-Pax and p-FAK in a punctuated pattern (white arrows). DIC: Differential interference contrast microscopy. Pax: paxillin. A488: Alexa Fluor 488. The bars represent 5 μm.

Article Snippet: Expression of C3G, Abi1 and Cbl was silenced in K562 cells by transfection of (h) Lentiviral Particles: C3G shRNA (sc-29863-V), Abi1 shRNA (sc-40306-V), Cbl shRNA (sc-29242-V) and control shRNA (sc-108080) from Santa Cruz Biotechnology, following the manufacturer`s protocol.

Techniques: Immunoprecipitation, Cell Culture, Expressing, Western Blot, Confocal Microscopy, Labeling, Control, Incubation, Microscopy

Journal: Cell Communication and Signaling

Article Title: C3G forms complexes with Bcr-Abl and p38α MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion

doi: 10.1186/1478-811x-11-9

Figure Lengend Snippet: Figure 8 Abi1, Cbl and p130Cas contribute to FA regulation. (A) Representative Western blots showing the expression and phosphorylation (p) of the indicated proteins in K562 cells upon knock-down of Cbl (clone 1), Abi1 (clone 2) or p130Cas (clone 5), and their corresponding control cells (see Figure 3). ß-actin was used as loading control. Relative ratios between the levels of the different analyzed proteins and ß-actin are shown. (B) The histograms represent the relative values of the levels of expression of the analyzed proteins relative to ß-tubulin. shCT-sc: Control shRNA from Santa Cruz Biotech., shCT-sg: Control shRNA from Sigma. Susp: suspension; FN: fibronectin.

Article Snippet: Expression of C3G, Abi1 and Cbl was silenced in K562 cells by transfection of (h) Lentiviral Particles: C3G shRNA (sc-29863-V), Abi1 shRNA (sc-40306-V), Cbl shRNA (sc-29242-V) and control shRNA (sc-108080) from Santa Cruz Biotechnology, following the manufacturer`s protocol.

Techniques: Western Blot, Expressing, Phospho-proteomics, Knockdown, Control, shRNA, Suspension