Journal: Cancer Cell International

Article Title: Knock-down of Kaiso induces proliferation and blocks granulocytic differentiation in blast crisis of chronic myeloid leukemia

doi: 10.1186/1475-2867-12-28

Figure Lengend Snippet: Kaiso subcellular localization in K562 cell line (A). Immunofluorescence staining of Kaiso . Kaiso was distributed in the cytoplasm, with less visible staining in the nucleus ( B ). Immunofluorescence of Kaiso in imatinib-resistant K562 cells and K562 cells treated with imatinib. ( C ). Expression analysis of Kaiso by immunoblotting assay. Normal bands can be detected after transfection with siRNA-p120ctn and treatment with imatinib. Little bands can be detected after transfection with siRNA-Kaiso and imatinib-resistant K562 cells. β-tubulin served as an internal control. ( D ). To verify effective separation of the cytoplasmic and nuclear fractions, cytoplasmic and nuclear extracts were immunoblotted for Kaiso in normal (K562) and imatinib-resistant K562 (IR-K562) cell line.

Article Snippet: Primary antibodies were the following: anti-Kaiso (mouse 1:100; Santa Cruz, CA), anti β-tubulin (mouse 1:500; Sigma), Secondary antibodies were incubated for 2 h at room temperature.

Techniques: Immunofluorescence, Staining, Expressing, Western Blot, Transfection

Journal: Cancer Cell International

Article Title: Knock-down of Kaiso induces proliferation and blocks granulocytic differentiation in blast crisis of chronic myeloid leukemia

doi: 10.1186/1475-2867-12-28

Figure Lengend Snippet: siRNA-Kaiso efficiently down-regulates cytoplasmic Kaiso expression in K562 cell line . ( A ). Immunofluorescence staining of Kaiso and β-tubulin. siRNA-Kaiso only efficiently downregulated cytoplasmic Kaiso expression in k562 cell line when compared to the scrambled knock-down cells ( B ). Cytoplasmic vs. nuclear fractionation Western blot analysis confirmed the results of immunofluorescence staining. β-tubulin served as an internal control. ( C ). Expression analysis of Kaiso knock-down by Real Time RT-PCR. ( D ). Expression analysis of p120ctn knock-down by Real Time RT-PCR. Data were expressed as mean ± standard deviation (S.D.). Columns, mean (n = 3); error bars, S.D.

Article Snippet: Primary antibodies were the following: anti-Kaiso (mouse 1:100; Santa Cruz, CA), anti β-tubulin (mouse 1:500; Sigma), Secondary antibodies were incubated for 2 h at room temperature.

Techniques: Expressing, Immunofluorescence, Staining, Fractionation, Western Blot, Quantitative RT-PCR, Standard Deviation

Journal: Cancer Cell International

Article Title: Knock-down of Kaiso induces proliferation and blocks granulocytic differentiation in blast crisis of chronic myeloid leukemia

doi: 10.1186/1475-2867-12-28

Figure Lengend Snippet: Expression analysis of Wnt11, β-catenin, Kaiso and p120ctn genes in either Kaiso (A) or p120ctn (B) alone or in combination (C) knock-down cells . K562 cells were transfected with the indicated siRNA combinations. Twenty-four hours later, RNA was isolated and subjected to Real Time RT-PCR to quantify expression of Wnt11, β-catenin, Kaiso and p120ctn after normalization to β-actin and compared to the scrambled knock-down cells. Data were expressed as mean ± standard deviation (S.D.). Columns, mean (n = 3); error bars, S.D.; *, p < 0.001.

Article Snippet: Primary antibodies were the following: anti-Kaiso (mouse 1:100; Santa Cruz, CA), anti β-tubulin (mouse 1:500; Sigma), Secondary antibodies were incubated for 2 h at room temperature.

Techniques: Expressing, Transfection, Isolation, Quantitative RT-PCR, Standard Deviation

Journal: Cancer Cell International

Article Title: Knock-down of Kaiso induces proliferation and blocks granulocytic differentiation in blast crisis of chronic myeloid leukemia

doi: 10.1186/1475-2867-12-28

Figure Lengend Snippet: Kaiso knock-down inhibits cell differentiation . K562 cells were transfected with the indicated siRNA combinations. Twenty-four hours later, RNA was isolated and subjected to Real Time RT-PCR to quantify expression of C-MyB, PU-1, C/EBPalpha and Gata-2 after normalization to β-actin and compared to the scrambled knock-down cells. Data were expressed as mean ± standard deviation (S.D.). Columns, mean (n = 3); error bars, S.D.; *, p < 0.001.

Article Snippet: Primary antibodies were the following: anti-Kaiso (mouse 1:100; Santa Cruz, CA), anti β-tubulin (mouse 1:500; Sigma), Secondary antibodies were incubated for 2 h at room temperature.

Techniques: Cell Differentiation, Transfection, Isolation, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: Cancer Cell International

Article Title: Knock-down of Kaiso induces proliferation and blocks granulocytic differentiation in blast crisis of chronic myeloid leukemia

doi: 10.1186/1475-2867-12-28

Figure Lengend Snippet: Effect of RNAi knock-down of Kaiso on LAMA-84. ( A ) Kaiso knock-down induce Wnt11 expression. LAMA-84 cells were transfected with 20nM of siRNA. Twenty-four hours later, RNA was isolated and subjected to Real Time RT-PCR to quantify expression of Wnt11, β-catenin, Kaiso and p120ctn after normalization to β-actin and compared to the scrambled knock-down cells. Columns, mean (n= 2). ( B ) Kaiso knock-down improve SCF expression in LAMA-84. Expression analysis of SCF gene by Real Time RT-PCR in Kaiso knock-down cells. Columns, mean (n= 2). ( C ) Effect of Kaiso knock-down on cell differentiation. LAMA-84 cells were transfected with 20nM of siRNA. Twenty-four hours later, RNA was isolated and subjected to Real Time RT-PCR to quantify expression of C-MyB, PU-1, C/EBPalpha and Gata-2 after normalization to β-actin and compared to the scrambled knock-down cells. Columns, mean (n= 2). ( D ) Knocking-down expression of Kaiso in LAMA-84 cells induced monocyte-macrophage differentiation. Flow cytometric measurement of granulocytic specific surface antigen CD15 and CD11b in LAMA-84 cells after normalization with the scrambled knock-down expression. LAMA cells were transfected with 20nM of siRNA after 50 hr-incubation. The CD117 (c-Kit receptor), CD33 and the erythroid markers GPA and CD71 were also analyzed. Columns, mean (n= 2).

Article Snippet: Primary antibodies were the following: anti-Kaiso (mouse 1:100; Santa Cruz, CA), anti β-tubulin (mouse 1:500; Sigma), Secondary antibodies were incubated for 2 h at room temperature.

Techniques: Expressing, Transfection, Isolation, Quantitative RT-PCR, Cell Differentiation, Incubation

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Jinfu’an Decoction Inhibits Invasion and Metastasis in Human Lung Cancer Cells (H1650) via p120ctn-Mediated Induction and Kaiso

doi: 10.12659/MSM.909748

Figure Lengend Snippet: P120ctn in lung cancer cell H1650 in the blank control group was mainly located and significantly overexpressed in the cytoplasm and cytomembrane. Moreover, cultured with JFA decoction, its expression level was decreased ( A ). In contrast, Kaiso in the blank control group was more often located in cytoplasm and in the JFA decoction group its level was increased ( B ). Immunofluorescence staining revealed that expression of p120ctn with the JFA decoction was downregulated, but the protein level of Kaiso was upregulated ( C ). The scale is 50 μm in each image. ** p<0.05 compared with the blank control group.

Article Snippet: When reaching 60% confluence, cells treated with indicated concentrations were fixed with 4% methanol, permeabilized with 0.1% Triton-X-100, and blocked with 5% BSA for 1 h, before being incubated at 4°C overnight with anti-p120ctn mAb (BD Transduction Laboratories) and anti-Kaiso mAb (Santa Cruz Biotechnology, INC), followed by incubation with a secondary antibody conjugated to TRITC/FITC-labeled antibody.

Techniques: Cell Culture, Expressing, Immunofluorescence, Staining

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Jinfu’an Decoction Inhibits Invasion and Metastasis in Human Lung Cancer Cells (H1650) via p120ctn-Mediated Induction and Kaiso

doi: 10.12659/MSM.909748

Figure Lengend Snippet: JFA decoction decreased the expression of p120ctn, its isoform 1A, and p120ctn S288 phosphorylation compared with the blank control group but enhanced the protein expression of Kaiso. ** p<0.05 compared with the blank control group.

Article Snippet: When reaching 60% confluence, cells treated with indicated concentrations were fixed with 4% methanol, permeabilized with 0.1% Triton-X-100, and blocked with 5% BSA for 1 h, before being incubated at 4°C overnight with anti-p120ctn mAb (BD Transduction Laboratories) and anti-Kaiso mAb (Santa Cruz Biotechnology, INC), followed by incubation with a secondary antibody conjugated to TRITC/FITC-labeled antibody.

Techniques: Expressing

Journal: bioRxiv

Article Title: T606-phosphorylation deprives the function of Kaiso as a transcription and oncogenic factor

doi: 10.1101/2020.03.23.003509

Figure Lengend Snippet: Different phosphorylation states of Kaiso protein in the cytoplasm and nucleus of cells in vitro and in vivo . ( A ) The phosphorylation statuses of GFP-Kaiso stably transfected MGC803 cells and the corresponding xenograft tissues in the Phos-tag SDS-PAGE analysis. ( B ) The phosphorylation statuses of endogenous cytoplasmic and nuclear Kaiso in MGC803 cells were validated using anti-phosphoSer/Thr/Tyr universal antibody. ( C ) Subcellular location of endogenous pT606-Kaiso and total Kaiso in cytoplasmic and nuclear proteins (with and without de-phosphorylation treatment by CIAP for 30 min) extracted from 3 cancer cell lines in Western blotting. ( D ) Locations of pT606-Kaiso and total Kaiso in the cytoplasm and nucleus in an indirect immunofluorescence confocal analysis. ( E ) Locations of pT606-Kaiso and total Kaiso in a representative gastric carcinoma and the paired normal tissues in IHC analysis. ( F ) Locations of pT606-Kaiso and total Kaiso in human gastric tissues in the indirect immunofluorescence confocal analysis.

Article Snippet: Rabbit pT606-Kaiso polyclonal antibody (1:100) and mouse monoclonal antibody for total Kaiso (1:50, sc-23871, Santa Cruz) were used in the IHC analysis.

Techniques: In Vitro, In Vivo, Stable Transfection, Transfection, SDS Page, De-Phosphorylation Assay, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: T606-phosphorylation deprives the function of Kaiso as a transcription and oncogenic factor

doi: 10.1101/2020.03.23.003509

Figure Lengend Snippet: Characterizing the specificity of pT606-Kaiso polyclonal antibody. ELISA results for the specificity of pT606-Kaiso and control antibodies against the pT606-Kaiso peptide (LSDRSS pT IPAM, the top chart) and for the specificity of pT606-Kaiso and control antibodies for the nonphosphorylated control peptide (LSDRSS T IPAM, the bottom chart).

Article Snippet: Rabbit pT606-Kaiso polyclonal antibody (1:100) and mouse monoclonal antibody for total Kaiso (1:50, sc-23871, Santa Cruz) were used in the IHC analysis.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: T606-phosphorylation deprives the function of Kaiso as a transcription and oncogenic factor

doi: 10.1101/2020.03.23.003509

Figure Lengend Snippet: Kaiso interacted with 14-3-3 family members depending on Kaiso pT606. ( A ) After the plasmids of 14-3-3 family members and GST-Kaiso were co-transfected into MGC803 cells, GST-Kaiso pulled down various isoforms of 14-3-3 family, especially 14-3-3σ. ( B ) Endogenous Kaiso immunoprecipitated pan-14-3-3 or 14-3-3σ protein immunoprecipitated endogenous Kaiso in MGC803 cell in Co-IP analysis. ( C ) T606A mutation of Kaiso concealed its interaction with pan-14-3-3 in MGC803 cells in Co-IP analysis.

Article Snippet: Rabbit pT606-Kaiso polyclonal antibody (1:100) and mouse monoclonal antibody for total Kaiso (1:50, sc-23871, Santa Cruz) were used in the IHC analysis.

Techniques: Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Mutagenesis

Journal: bioRxiv

Article Title: T606-phosphorylation deprives the function of Kaiso as a transcription and oncogenic factor

doi: 10.1101/2020.03.23.003509

Figure Lengend Snippet: Effects of 14-3-3σ on inhibition of Kaiso’s target gene CDH1 expression. ( A ) The Pearson correlation coefficient between mRNA levels of 14-3-3 family members and Kaisos’ target genes in public RNA-seq datasets from Cancer Cell Line Encyclopedia (CCLE, 1156 cancer cell lines) and Genotype-Tissue Expression (GTEx, 11688 human normal tissues). ( B ) The level of CDH1 in SGC7901 and MKN45 cells with or without wild type Kaiso and 14-3-3σ overexpression. ( C ) The level of the CDH1 mRNA in SGC7901 and MKN45 cells with or without wild type Kaiso and 14-3-3σ overexpression ( D ) The amounts of pT606-Kaiso, total Kaiso, 14-3-3 σ, and CDH1 proteins in gastric carcinoma (T) and the paired normal tissues (N) from 12 patients by Western blotting. ( E ) Correlation between ratios of pT606-Kaiso to total Kaiso and CDH1 to GAPDH proteins based on the density of these proteins in Western blot.

Article Snippet: Rabbit pT606-Kaiso polyclonal antibody (1:100) and mouse monoclonal antibody for total Kaiso (1:50, sc-23871, Santa Cruz) were used in the IHC analysis.

Techniques: Inhibition, Expressing, RNA Sequencing Assay, Over Expression, Western Blot

Journal: bioRxiv

Article Title: T606-phosphorylation deprives the function of Kaiso as a transcription and oncogenic factor

doi: 10.1101/2020.03.23.003509

Figure Lengend Snippet: The status of Kaiso structure and protein modifications detected by LC/MS. Phosphorylation at the Thr606 residue in the LSDRSS T IPAM motif was illustrated in the bottom chart in details. The image was adapted with graphs for Kaiso modifications from the web site ( www.phosphosite.org ) ( , ). AlphaFold predicted 3D structures for Kaiso (ZBTB33/Q86T24) protein was adapted from images downloaded from the website ( https://alphafold.ebi.ac.uk ) ; pLDDT, AlphaFold produced per-residue confidence score between 0 and 100. Information for Kaiso domains was adapted from images downloaded from the website ( https://www.uniprot.org/uniprot/Q86T24 ) ( , ).

Article Snippet: Antibodies for Kaiso (sc-365428, Santa Cruz, USA), P120ctn (66208-1, Proteintech, IL, USA), pan-14-3-3 (sc-629, Santa Cruz), 14-3-3 family (α/β, ε, η, δ/γ, τ, ζ, σ) kits (#9769, CST, USA), 14-3-3σ (sc-100638, Santa Cruz), Ser/Thr/Tyr phosphor-protein (ab15556, Abcam, UK), phosphorylated AKT substrate [(R/K)X(R/K)XX(pT/pS)] (pAKT-sub; #9611, Cell Signaling Technology), CDH1 (#3195, CST), LAMIN B1 (66095-1, Proteintech), β-TUBLIN (66240-1, Proteintech), HA (M20003, Abmart, Shanghai, China), FLAG (66008-2, Proteintech), GFP (NB100-1614, Novus, CO, USA), GST (66001-1, Proteintech), GAPDH (660004-1, Proteintech) were used in the IP and Western blotting analyses.

Techniques: Liquid Chromatography with Mass Spectroscopy, Produced

Journal: bioRxiv

Article Title: T606-phosphorylation deprives the function of Kaiso as a transcription and oncogenic factor

doi: 10.1101/2020.03.23.003509

Figure Lengend Snippet: Different phosphorylation states of Kaiso protein in the cytoplasm and nucleus of cells in vitro and in vivo . ( A ) The phosphorylation statuses of GFP-Kaiso stably transfected MGC803 cells and the corresponding xenograft tissues in the Phos-tag SDS-PAGE analysis. ( B ) The phosphorylation statuses of endogenous cytoplasmic and nuclear Kaiso in MGC803 cells were validated using anti-phosphoSer/Thr/Tyr universal antibody. ( C ) Subcellular location of endogenous pT606-Kaiso and total Kaiso in cytoplasmic and nuclear proteins (with and without de-phosphorylation treatment by CIAP for 30 min) extracted from 3 cancer cell lines in Western blotting. ( D ) Locations of pT606-Kaiso and total Kaiso in the cytoplasm and nucleus in an indirect immunofluorescence confocal analysis. ( E ) Locations of pT606-Kaiso and total Kaiso in a representative gastric carcinoma and the paired normal tissues in IHC analysis. ( F ) Locations of pT606-Kaiso and total Kaiso in human gastric tissues in the indirect immunofluorescence confocal analysis.

Article Snippet: Antibodies for Kaiso (sc-365428, Santa Cruz, USA), P120ctn (66208-1, Proteintech, IL, USA), pan-14-3-3 (sc-629, Santa Cruz), 14-3-3 family (α/β, ε, η, δ/γ, τ, ζ, σ) kits (#9769, CST, USA), 14-3-3σ (sc-100638, Santa Cruz), Ser/Thr/Tyr phosphor-protein (ab15556, Abcam, UK), phosphorylated AKT substrate [(R/K)X(R/K)XX(pT/pS)] (pAKT-sub; #9611, Cell Signaling Technology), CDH1 (#3195, CST), LAMIN B1 (66095-1, Proteintech), β-TUBLIN (66240-1, Proteintech), HA (M20003, Abmart, Shanghai, China), FLAG (66008-2, Proteintech), GFP (NB100-1614, Novus, CO, USA), GST (66001-1, Proteintech), GAPDH (660004-1, Proteintech) were used in the IP and Western blotting analyses.

Techniques: In Vitro, In Vivo, Stable Transfection, Transfection, SDS Page, De-Phosphorylation Assay, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: T606-phosphorylation deprives the function of Kaiso as a transcription and oncogenic factor

doi: 10.1101/2020.03.23.003509

Figure Lengend Snippet: AKT1 increases the phosphorylation of Kaiso at T606. (A ) A conservative RSX T XP motif within Kaiso of human and other species ( B ) After starvation overnight, treatments of Insulin (100 ng/mL), IL-6 (10 ng/mL) and fetal bovine serum (FBS, 1:10000 v/v) for 15 min increased the phosphorylation level of endogenous Kaiso in MGC803 cells. ( C ) Effects of AKT inhibitor MK2206 treatment (10 μmol/mL) for 30 min blocked the promotion of Insulin induced Kaiso phosphorylation as pAKT substrate in MGC803 cells. ( D ) AKT1 overexpression at different doses increased the amount of phosphorylated AKT substrate (pAKT-sub) in Kaiso complexes immunoprecipitated by Kaiso antibody in MGC803 cells. ( E ) The activity comparison of three kinase candidates to phosphorylate Kaiso at T606 in MGC803 and BGC823 cells. ( F ) The T606-phosphorylation status of endogenous Kaiso in MGC803 and BGC823 with AKT1 overexpression after starvation overnight.

Article Snippet: Antibodies for Kaiso (sc-365428, Santa Cruz, USA), P120ctn (66208-1, Proteintech, IL, USA), pan-14-3-3 (sc-629, Santa Cruz), 14-3-3 family (α/β, ε, η, δ/γ, τ, ζ, σ) kits (#9769, CST, USA), 14-3-3σ (sc-100638, Santa Cruz), Ser/Thr/Tyr phosphor-protein (ab15556, Abcam, UK), phosphorylated AKT substrate [(R/K)X(R/K)XX(pT/pS)] (pAKT-sub; #9611, Cell Signaling Technology), CDH1 (#3195, CST), LAMIN B1 (66095-1, Proteintech), β-TUBLIN (66240-1, Proteintech), HA (M20003, Abmart, Shanghai, China), FLAG (66008-2, Proteintech), GFP (NB100-1614, Novus, CO, USA), GST (66001-1, Proteintech), GAPDH (660004-1, Proteintech) were used in the IP and Western blotting analyses.

Techniques: Over Expression, Immunoprecipitation, Activity Assay

Journal: bioRxiv

Article Title: T606-phosphorylation deprives the function of Kaiso as a transcription and oncogenic factor

doi: 10.1101/2020.03.23.003509

Figure Lengend Snippet: AKT1 and 14-3-3 regulate the T606-phosphorylation and subcellular localization of endogenous Kaiso. (A ) Endogenous Kaiso in MGC803 cells immunoprecipitated by Kaiso antibody was identified by the antibody specific for AKT substrate motif, and the immunoprecipitation by AKT substrate antibody was identified by antibody against Kaiso. ( B ) AKT1 overexpression increased endogenous Kaiso-14-3-3 interaction in MGC803 cells in Co-IP assay. ( C and D ) The T606-phosphorylation states of endogenous Kaiso in the cytoplasm and nucleus of MGC803 and BGC823 cells with 14-3-3σ and 14-3-3γ overexpression, respectively.

Article Snippet: Antibodies for Kaiso (sc-365428, Santa Cruz, USA), P120ctn (66208-1, Proteintech, IL, USA), pan-14-3-3 (sc-629, Santa Cruz), 14-3-3 family (α/β, ε, η, δ/γ, τ, ζ, σ) kits (#9769, CST, USA), 14-3-3σ (sc-100638, Santa Cruz), Ser/Thr/Tyr phosphor-protein (ab15556, Abcam, UK), phosphorylated AKT substrate [(R/K)X(R/K)XX(pT/pS)] (pAKT-sub; #9611, Cell Signaling Technology), CDH1 (#3195, CST), LAMIN B1 (66095-1, Proteintech), β-TUBLIN (66240-1, Proteintech), HA (M20003, Abmart, Shanghai, China), FLAG (66008-2, Proteintech), GFP (NB100-1614, Novus, CO, USA), GST (66001-1, Proteintech), GAPDH (660004-1, Proteintech) were used in the IP and Western blotting analyses.

Techniques: Immunoprecipitation, Over Expression, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: T606-phosphorylation deprives the function of Kaiso as a transcription and oncogenic factor

doi: 10.1101/2020.03.23.003509

Figure Lengend Snippet: Characterizing the specificity of pT606-Kaiso polyclonal antibody. ELISA results for the specificity of pT606-Kaiso and control antibodies against the pT606-Kaiso peptide (LSDRSS pT IPAM, the top chart) and for the specificity of pT606-Kaiso and control antibodies for the nonphosphorylated control peptide (LSDRSS T IPAM, the bottom chart).

Article Snippet: Antibodies for Kaiso (sc-365428, Santa Cruz, USA), P120ctn (66208-1, Proteintech, IL, USA), pan-14-3-3 (sc-629, Santa Cruz), 14-3-3 family (α/β, ε, η, δ/γ, τ, ζ, σ) kits (#9769, CST, USA), 14-3-3σ (sc-100638, Santa Cruz), Ser/Thr/Tyr phosphor-protein (ab15556, Abcam, UK), phosphorylated AKT substrate [(R/K)X(R/K)XX(pT/pS)] (pAKT-sub; #9611, Cell Signaling Technology), CDH1 (#3195, CST), LAMIN B1 (66095-1, Proteintech), β-TUBLIN (66240-1, Proteintech), HA (M20003, Abmart, Shanghai, China), FLAG (66008-2, Proteintech), GFP (NB100-1614, Novus, CO, USA), GST (66001-1, Proteintech), GAPDH (660004-1, Proteintech) were used in the IP and Western blotting analyses.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: T606-phosphorylation deprives the function of Kaiso as a transcription and oncogenic factor

doi: 10.1101/2020.03.23.003509

Figure Lengend Snippet: 14-3-3σ promotes the interaction of Kaiso and p120ctn in the cytoplasm. ( A ) More Kaiso-p120 complex was immunoprecipitated by Kaiso antibody in MGC803 cells with 14-3-3σ overexpression in Co-IP assay. Total protein levels of p120ctn and 14-3-3σ in the lysate of MGC803 cells with 14-3-3σ overexpression were displayed in the right side. ( B ) Alterations of interactions between Kaiso and p120ctn proteins in the cytoplasm and nucleus of these cells with 14-3-3σ overexpression in the Co-IP assay using Kaiso antibody. ( C ) The subcellular locations of Kaiso, p120ctn, and mCherry-14-3-3σ in MGC803 cells. ( D ) Western blotting images for detecting effect of p120ctn knockdown and 14-3-3σ overexpression on distribution of Kaiso in the cytoplasm and nucleus of MGC803 cells. ( E ) The levels of total p120ctn and Kaiso proteins in the lysate of MGC803 cells with 14-3-3σ overexpression and siRNAs knockdown of p120ctn expression.

Article Snippet: Antibodies for Kaiso (sc-365428, Santa Cruz, USA), P120ctn (66208-1, Proteintech, IL, USA), pan-14-3-3 (sc-629, Santa Cruz), 14-3-3 family (α/β, ε, η, δ/γ, τ, ζ, σ) kits (#9769, CST, USA), 14-3-3σ (sc-100638, Santa Cruz), Ser/Thr/Tyr phosphor-protein (ab15556, Abcam, UK), phosphorylated AKT substrate [(R/K)X(R/K)XX(pT/pS)] (pAKT-sub; #9611, Cell Signaling Technology), CDH1 (#3195, CST), LAMIN B1 (66095-1, Proteintech), β-TUBLIN (66240-1, Proteintech), HA (M20003, Abmart, Shanghai, China), FLAG (66008-2, Proteintech), GFP (NB100-1614, Novus, CO, USA), GST (66001-1, Proteintech), GAPDH (660004-1, Proteintech) were used in the IP and Western blotting analyses.

Techniques: Immunoprecipitation, Over Expression, Co-Immunoprecipitation Assay, Western Blot, Expressing