Journal: International Journal of Molecular Sciences

Article Title: p53, miR-34a and EMP1—Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

doi: 10.3390/ijms20174129

Figure Lengend Snippet: TFF3 overexpression enhances p53 transcriptional activity and protein expression and increases miR-34a expression with downstream reduction of EMP1 in Y79 RB cells. ( A ) Quantitative Real-time PCR confirmation of TFF3 lentiviral overexpression (Trefoil factor family peptide 3 (TFF3)) in Y79 cells compared to control cells (ctr). ( B ) Luciferase assays were performed with Y79 cells transiently transfected with TFF3 or empty vector control (ctr) in addition to wild-type PG13-Luc (wt PG13) or mutant MG15-Luc (mut MG15). Forced TFF3 expression leads to an increased luciferase signal upon p53 promotor activation in Y79 cells. ( C ) Western blot analysis showing increased p53 and TFF3 protein levels after TFF3 overexpression (TFF3). The indicated intensity ratios of p53 and TFF3 protein levels relative to β-actin levels were calculated using ImageJ software. ( D , E ) Quantitative real-time PCR analysis of miR-34a and EMP1 expression levels in Y79 cells compared to control cells after lentiviral TFF3 overexpression (ctr). Values are means of at least 3 independent experiments ± SEM. * p -value < 0.05, ** p -value < 0.01; statistical differences compared to the control group calculated by Student’s t -test or one-way ANOVA and Newman-Keuls post test.

Article Snippet: To generate a lentiviral EMP1 expression vector (pLenti CMV_ EMP1 ), the human EMP1 cDNA sequence was amplified from the RC208410 vector (Origene) using the forward primer 5′-C GAATTC TATGTTGGTATTGCTGGCTGG-3′ and the reverse primer 5′-CG CTCGAG TTATTTCTTTCTCAGGACCAG-3′ containing Eco RI and Xho I restriction sites (underlined).

Techniques: Over Expression, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Transfection, Plasmid Preparation, Mutagenesis, Activation Assay, Western Blot, Software

Journal: International Journal of Molecular Sciences

Article Title: p53, miR-34a and EMP1—Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

doi: 10.3390/ijms20174129

Figure Lengend Snippet: Epithelial membrane protein 1 (EMP1) knockdown leads to reduced cell viability and proliferation and induces apoptosis in Y79 RB cells. ( A ) Western blot data confirmed decreased EMP1 protein levels after EMP1 knockdown (shEMP1) in Y79 cells. The CML cell line K562 served as an EMP1 positive control, ß-actin as a loading control. ( B , C ) Stably EMP1 knockdown Y79 RB cells (shEMP1) showed significantly decreased viability and proliferation levels compared to control cells (ctr) as revealed by ( B ) WST-1 assays and ( C ) BrdU stains. ( D ) EMP1 knockdown Y79 cells (shEMP1) displayed a significantly increased apoptosis rate compared to control cells (ctr) as revealed by DAPI stains. Values are means of 3 independent experiments ± SEM. *** p -value < 0.001 statistical differences compared to the control group calculated by Student’s t -test.

Article Snippet: To generate a lentiviral EMP1 expression vector (pLenti CMV_ EMP1 ), the human EMP1 cDNA sequence was amplified from the RC208410 vector (Origene) using the forward primer 5′-C GAATTC TATGTTGGTATTGCTGGCTGG-3′ and the reverse primer 5′-CG CTCGAG TTATTTCTTTCTCAGGACCAG-3′ containing Eco RI and Xho I restriction sites (underlined).

Techniques: Western Blot, Positive Control, Stable Transfection

Journal: International Journal of Molecular Sciences

Article Title: p53, miR-34a and EMP1—Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

doi: 10.3390/ijms20174129

Figure Lengend Snippet: EMP1 overexpression leads to increased cell viability and proliferation and induces caspase-3/7 dependent apoptosis in Y79 RB cells. ( A ) Western blot data confirmed increased EMP1 protein levels after EMP1 overexpression (EMP1) in Y79 cells. The CML cell line K562 served as an EMP1 positive control, ß-actin as a loading control. ( B , C ) Stably EMP1 overexpressing Y79 RB cells (EMP1) showed significantly increased viability and proliferation levels compared to control cells (ctr) as revealed by ( B ) WST-1 assays and ( C ) BrdU stains. red: BrdU-labeled cells; blue: DAPI counterstaining ( D ) Growth curve analysis of EMP1 overexpressing Y79 RB cells showed a significant increase in cell growth rates. ( E ) EMP1 overexpressing Y79 cells (EMP1) displayed a significantly reduced apoptosis rate compared to control cells (ctr) as revealed by DAPI stains. ( F ) Caspase-3/7 activity was significantly reduced after EMP1 overexpression in Y79 RB cells (EMP1) compared to control cells (ctr). Values are means of at least 3 independent experiments ± SEM. ** p -value < 0.01; *** p -value < 0.001 statistical differences compared to the control group calculated by Student’s t -test.

Article Snippet: To generate a lentiviral EMP1 expression vector (pLenti CMV_ EMP1 ), the human EMP1 cDNA sequence was amplified from the RC208410 vector (Origene) using the forward primer 5′-C GAATTC TATGTTGGTATTGCTGGCTGG-3′ and the reverse primer 5′-CG CTCGAG TTATTTCTTTCTCAGGACCAG-3′ containing Eco RI and Xho I restriction sites (underlined).

Techniques: Over Expression, Western Blot, Positive Control, Stable Transfection, Labeling, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: p53, miR-34a and EMP1—Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

doi: 10.3390/ijms20174129

Figure Lengend Snippet: Effect of stable EMP1 overexpression on RB cell colony formation and anchorage independent growth. ( A ) Quantification of colony formation assays (CFA) showing a significant higher capacity of EMP1 overexpressing Y79 RB cells to form colonies (EMP1) compared to control cells (ctr). ( B ) Quantification of anchorage independent growth capacity of EMP1 overexpressing Y79 RB cells (EMP1) compared to control cells (ctr) as revealed by soft agarose assay. All photographs are taken 3 weeks after seeding EMP1 overexpressing and control Y79 RB cells. Values are means of at least 3 independent experiments ± SEM. *** p -value < 0.001; statistical differences compared to the control group calculated by Student’s t -test.

Article Snippet: To generate a lentiviral EMP1 expression vector (pLenti CMV_ EMP1 ), the human EMP1 cDNA sequence was amplified from the RC208410 vector (Origene) using the forward primer 5′-C GAATTC TATGTTGGTATTGCTGGCTGG-3′ and the reverse primer 5′-CG CTCGAG TTATTTCTTTCTCAGGACCAG-3′ containing Eco RI and Xho I restriction sites (underlined).

Techniques: Over Expression

Journal: International Journal of Molecular Sciences

Article Title: p53, miR-34a and EMP1—Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

doi: 10.3390/ijms20174129

Figure Lengend Snippet: Stable, lentiviral EMP1 overexpression increases tumor formation capacity of Y79 RB cells. ( A ) Photographs of CAM tumors in situ (left column), 3D tumor volume (middle column) and ruler measurements (in cm) of excised tumors (right column) revealing that tumors forming in the upper CAM after grafting EMP1 overexpressing Y79 RB cells were significantly larger compared to those developing from control cells (ctr). ( B , C ) Quantification of CAM assays by ( B ) tumor size, ( C ) tumor weight and ( D ) tumor volume. Values are means from at least 3 independent experiments (except for tumor volume which was measured exemplarily in one experimental setting) ± SEM. * p -value < 0.05, ** p -value < 0.01 statistical differences compared to the control group calculated by Student’s t -test.

Article Snippet: To generate a lentiviral EMP1 expression vector (pLenti CMV_ EMP1 ), the human EMP1 cDNA sequence was amplified from the RC208410 vector (Origene) using the forward primer 5′-C GAATTC TATGTTGGTATTGCTGGCTGG-3′ and the reverse primer 5′-CG CTCGAG TTATTTCTTTCTCAGGACCAG-3′ containing Eco RI and Xho I restriction sites (underlined).

Techniques: Over Expression, In Situ

Journal: International Journal of Molecular Sciences

Article Title: p53, miR-34a and EMP1—Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

doi: 10.3390/ijms20174129

Figure Lengend Snippet: MiR-34a and EMP1 overexpression leads to enhanced chemosensitivity. ( A ) Overexpression of miR-34a in Y79 RB cells leads to significantly increased apoptosis levels compared to control cells (ctr). Additional treatment with etoposide (Etop), cisplatin (CisP), or vincristine (Vin) significantly elevates the apoptosis levels compared to untreated miR-34a overexpressing Y79 RB cells. ( B ) Overexpression of EMP1 in Y79 RB cells leads to significantly decreased apoptosis levels compared to control cells (ctr). Additional treatment with etoposide (Etop) and vincristine (Vin) leads to higher apoptosis levels compared to untreated EMP1 overexpressing Y79 RB cells. Additional treatment with cisplatin (CisP) did not change the apoptosis level compared to untreated EMP1 expressing Y79 RB cells. Values are means of at least 3 independent experiments ± SEM. * p -value < 0.05, *** p -value < 0.001; statistical differences compared to the control group calculated by one-way ANOVA and Newman-Keuls post test.

Article Snippet: To generate a lentiviral EMP1 expression vector (pLenti CMV_ EMP1 ), the human EMP1 cDNA sequence was amplified from the RC208410 vector (Origene) using the forward primer 5′-C GAATTC TATGTTGGTATTGCTGGCTGG-3′ and the reverse primer 5′-CG CTCGAG TTATTTCTTTCTCAGGACCAG-3′ containing Eco RI and Xho I restriction sites (underlined).

Techniques: Over Expression, Expressing

Journal: Molecular Biology of the Cell

Article Title: p23/Tmp21 Differentially Targets the Rac-GAP β2-Chimaerin and Protein Kinase C via Their C1 Domains

doi: 10.1091/mbc.E09-08-0735

Figure Lengend Snippet: Differential interaction of C1 domains with p23/Tmp21. (A) Schematic representation of C1εa-b, C1εa, C1εb, C1ζ, or C1β2-ch domain fused to pLexA. (B) EGY48 yeast (containing 8op-LacZ vector) was cotransformed with pLexA encoding C1εa-b, C1εa, C1εb, C1ζ, or C1β2-ch domain, and pB42AD-HA-tagged p23/Tmp21 (aa 108-208). Assay of β-galactosidase activity on induction (top) or no-induction (bottom) plates was carried out 72 h after transformation. Gal/Raf, galactosidase/raffinose. (C) Assay of β-galactosidase activity in liquid cultures using ONPG as a substrate. Results were expressed as mean ± SD (n = 3). (D) GFP-PKCεC1b domain localizes in the perinuclear region. HeLa cells were transfected with pEGFP-C1εa, C1εb, C1ζ, or C1β2-ch. Forty-eight hours later, cells were fixed and localization examined by confocal microscopy. Bar, 10 μm. All experiments have been performed at least three times with similar results.

Article Snippet: The following primary antibodies were used: anti-pLexA (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β-actin (Sigma-Aldrich), anti-V5 (Invitrogen), anti-p23/Tmp21 (ProSci, Poway, CA), anti-GST, anti-hemagglutinin (HA), anti-green fluorescent protein (GFP) (Covance, Emeryville, CA), anti-Rac1 (Millipore, Billerica, MA), anti-PKCε (Cell Signaling Technology, Danvers, MA), and anti-GS28 (BD Biosciences, San Jose, CA).

Techniques: Plasmid Preparation, Activity Assay, Transformation Assay, Transfection, Confocal Microscopy

Journal: Molecular Biology of the Cell

Article Title: p23/Tmp21 Differentially Targets the Rac-GAP β2-Chimaerin and Protein Kinase C via Their C1 Domains

doi: 10.1091/mbc.E09-08-0735

Figure Lengend Snippet: Colocalization of GFP-fused PKCε C1b and β2-chimaerin C1 domains with p23/Tmp21. HeLa cells were cotransfected with pEGFP-fused C1εa, C1εb, C1ζ, or C1β2-ch (or empty vector) and V5-tagged full-length pcDNA3-p23/Tmp21. Forty-eight hours later, cells were fixed and stained with an anti-V5 antibody, and localization was examined by confocal microscopy. Colocalization images and Pearson's r (Rr) were generated by ImageJ. Similar results were observed at least in three independent experiments. Bar, 10 μm.

Article Snippet: The following primary antibodies were used: anti-pLexA (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β-actin (Sigma-Aldrich), anti-V5 (Invitrogen), anti-p23/Tmp21 (ProSci, Poway, CA), anti-GST, anti-hemagglutinin (HA), anti-green fluorescent protein (GFP) (Covance, Emeryville, CA), anti-Rac1 (Millipore, Billerica, MA), anti-PKCε (Cell Signaling Technology, Danvers, MA), and anti-GS28 (BD Biosciences, San Jose, CA).

Techniques: Plasmid Preparation, Staining, Confocal Microscopy, Generated

Journal: Molecular Biology of the Cell

Article Title: p23/Tmp21 Differentially Targets the Rac-GAP β2-Chimaerin and Protein Kinase C via Their C1 Domains

doi: 10.1091/mbc.E09-08-0735

Figure Lengend Snippet: Differential interaction of PKCε and β2-chimaerin with p23/Tmp21. (A and B) COS-1 cells were transfected with either pEBG (empty vector) or pEBG-p23/Tmp21. Twenty-four hours later, cells were infected with either HA-β2-chimaerin adenovirus (multiplicity of infection [MOI], 10 plaque-forming units [pfu]/cell) (A) or PKCε adenovirus (MOI = 3 pfu/cell) (B). After 24 h, cells were treated with PMA (1 μM) or vehicle for 30 min in the presence of the PKC inhibitor GF109203X (5 μM) and lysed. GST or GST-p23/Tmp21 proteins were precipitated with glutathione-Sepharose 4B beads, and associated HA-β2-chimaerin was detected by Western blot using an anti-HA antibody. Left, representative experiments. Right, densitometric analysis of three individual experiments, expressed as fold change relative to GST-p23/Tmp21 in the absence (A) or presence (B) of PMA. (C) HeLa cells were cotransfected with either pEGFP-PKCε or pEGFP-β2-chimaerin and pcDNA3.1/V5-p23/Tmp21 (full length). Forty-eight hours later, cells were treated with PMA (1 μM) or vehicle for 30 min, fixed, and stained with an anti-V5 antibody, and localization examined by confocal microscopy. Top, green fluorescence from GFP-PKCε or GFP-β2-chimaerin; middle, red fluorescence from pcDNA3.1/V5-p23/Tmp21; bottom, overlapped images. Colocalization images and Pearson's r (Rr) were generated by ImageJ. Similar results were obtained in three additional experiments. Bar, 10 μm.

Article Snippet: The following primary antibodies were used: anti-pLexA (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β-actin (Sigma-Aldrich), anti-V5 (Invitrogen), anti-p23/Tmp21 (ProSci, Poway, CA), anti-GST, anti-hemagglutinin (HA), anti-green fluorescent protein (GFP) (Covance, Emeryville, CA), anti-Rac1 (Millipore, Billerica, MA), anti-PKCε (Cell Signaling Technology, Danvers, MA), and anti-GS28 (BD Biosciences, San Jose, CA).

Techniques: Transfection, Plasmid Preparation, Infection, Western Blot, Staining, Confocal Microscopy, Fluorescence, Generated

Journal: Molecular Biology of the Cell

Article Title: p23/Tmp21 Differentially Targets the Rac-GAP β2-Chimaerin and Protein Kinase C via Their C1 Domains

doi: 10.1091/mbc.E09-08-0735

Figure Lengend Snippet: p23/Tmp21 RNAi depletion impairs perinuclear β2-chimaerin translocation. (A) Expression of p23/Tmp21 in HeLa cells stably expressing different p23/Tmp21 shRNAs (shRNA#1, shRNA#2, and shRNA#3) or control cells. (B) Cells were transfected with pEGFP-β2-chimaerin and 48 h later treated with PMA (3 μM) in the presence of GF109203X (5 μM). Time-lapse images of β2-chimaerin translocation in living cells were captured at different times after PMA treatment. Perinuclear and periphery translocation were marked with arrows. Similar results were observed in three individual experiments. Bar, 10 μm.

Article Snippet: The following primary antibodies were used: anti-pLexA (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β-actin (Sigma-Aldrich), anti-V5 (Invitrogen), anti-p23/Tmp21 (ProSci, Poway, CA), anti-GST, anti-hemagglutinin (HA), anti-green fluorescent protein (GFP) (Covance, Emeryville, CA), anti-Rac1 (Millipore, Billerica, MA), anti-PKCε (Cell Signaling Technology, Danvers, MA), and anti-GS28 (BD Biosciences, San Jose, CA).

Techniques: Translocation Assay, Expressing, Stable Transfection, shRNA, Control, Transfection

Journal: Molecular Biology of the Cell

Article Title: p23/Tmp21 Differentially Targets the Rac-GAP β2-Chimaerin and Protein Kinase C via Their C1 Domains

doi: 10.1091/mbc.E09-08-0735

Figure Lengend Snippet: Glu227 and Leu248 residues in the β2-chimaerin C1 domain are required for the interaction with p23/Tmp21. (A) EGY48 yeast (containing 8op-LacZ vector) was cotransformed with pLexA-β2-chim-C1 (E227G), pLexA-β2-chim-C1 (L248A), pLexA-β2-chim-C1 (E227G/L248A), or pLexA-β2-chim-C1 (C246A), and pB42AD-HA-tagged p23/Tmp21 (aa 108-208). Assay of β-galactosidase activity on induction (top) or no-induction (bottom) plates was carried out 72 h after transformation. Gal/Raf, galactosidase/raffinose. (B) COS-1 cells were cotransfected with either pEBG (empty vector) or pEBG-p23/Tmp21 and GFP vector, GFP-β2-chimaerin (wt), GFP-β2-chimaerin (E227G/L248A) or GFP-β2-chimaerin (C246A). After 24 h, cells were lysed. GST or GST-p23/Tmp21 proteins were precipitated with glutathione-Sepharose 4B beads and associated GFP-fused proteins detected by Western blot using an anti-GFP antibody. Left, representative experiments. Right, densitometric analysis of three individual experiments, expressed as fold change relative to GST-p23/Tmp21 bound β2-chimaerin (wt).

Article Snippet: The following primary antibodies were used: anti-pLexA (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β-actin (Sigma-Aldrich), anti-V5 (Invitrogen), anti-p23/Tmp21 (ProSci, Poway, CA), anti-GST, anti-hemagglutinin (HA), anti-green fluorescent protein (GFP) (Covance, Emeryville, CA), anti-Rac1 (Millipore, Billerica, MA), anti-PKCε (Cell Signaling Technology, Danvers, MA), and anti-GS28 (BD Biosciences, San Jose, CA).

Techniques: Plasmid Preparation, Activity Assay, Transformation Assay, Western Blot

Journal: Molecular Biology of the Cell

Article Title: p23/Tmp21 Differentially Targets the Rac-GAP β2-Chimaerin and Protein Kinase C via Their C1 Domains

doi: 10.1091/mbc.E09-08-0735

Figure Lengend Snippet: Colocalization studies of β2-chimaerin mutants and p23/Tmp-21. HeLa cells were cotransfected with pEGFP-β2-chim (wt), pEGFP-β2-chim (C246A), pEGFP-β2-chim (E227G), pEGFP-β2-chim (L248A), or pEGFP-β2-chim (E227G/L248A) and V5-tagged pcDNA-p23/Tmp21. Forty-eight hours later, cells were treated with PMA (3 μM) or vehicle for 30 min in the presence of GF 109203X (5 μM). Cells were then washed and visualized by confocal microscopy. Left, green fluorescence from GFP-β2-chimaerin (wild-type or mutants); middle, red fluorescence from pcDNA3.1/V5-p23/Tmp21; right, overlapped images. Far right, colocalization images generated by ImageJ. Similar results were obtained in three different experiments. Bar, 10 μm.

Article Snippet: The following primary antibodies were used: anti-pLexA (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β-actin (Sigma-Aldrich), anti-V5 (Invitrogen), anti-p23/Tmp21 (ProSci, Poway, CA), anti-GST, anti-hemagglutinin (HA), anti-green fluorescent protein (GFP) (Covance, Emeryville, CA), anti-Rac1 (Millipore, Billerica, MA), anti-PKCε (Cell Signaling Technology, Danvers, MA), and anti-GS28 (BD Biosciences, San Jose, CA).

Techniques: Confocal Microscopy, Fluorescence, Generated

Journal: Molecular Biology of the Cell

Article Title: p23/Tmp21 Differentially Targets the Rac-GAP β2-Chimaerin and Protein Kinase C via Their C1 Domains

doi: 10.1091/mbc.E09-08-0735

Figure Lengend Snippet: Identification of the β2-chimaerin C1 domain interacting region in p23/Tmp21. EGY48 yeast (containing 8op-LacZ vector) was cotransformed with pLexA-fused α1-chimaerin (aa 1–147) and pB42AD-HA–tagged p23/Tmp21 truncated mutants. (A) Top, schematic representation of p23/Tmp21 constructs used in the yeast two-hybrid assay. Middle, chimaerin expression in yeast lysates, as determined by Western blot using an anti-pLexA antibody; and expression of p23/Tmp21 truncated proteins in yeast lysates using an anti-HA antibody. Bottom, assay of β-galactosidase activity on induction or no-induction plates, carried out 72 h after transformation. Gal/Raf, galactosidase/raffinose. (B) COS-1 cells were transfected with either pEBG (empty vector) or pEBG-p23/Tmp21, and infected with a HA-β2-chimaerin adenovirus (multiplicity of infection [MOI] = 10 plaque-forming units [pfu]/cell). Thirty-six hours later, GST or GST-p23/Tmp21 proteins were precipitated with glutathione-Sepharose 4B beads and associated HA-β2-chimaerin detected by Western blot using an anti-HA antibody. Top, schematic representation of GST-p23/Tmp21 constructs used in the coprecipitation assays. Middle, expression of GST-p23/Tmp21 or its mutants and HA-β2-chimaerin in cell lysates. Bottom, associated HA-β2-chimaerin and GST-p23/Tmp21 or its mutants in pull-down assay were detected by Western blot using anti-HA and anti-GST antibody respectively. Similar results were observed in two additional experiments.

Article Snippet: The following primary antibodies were used: anti-pLexA (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β-actin (Sigma-Aldrich), anti-V5 (Invitrogen), anti-p23/Tmp21 (ProSci, Poway, CA), anti-GST, anti-hemagglutinin (HA), anti-green fluorescent protein (GFP) (Covance, Emeryville, CA), anti-Rac1 (Millipore, Billerica, MA), anti-PKCε (Cell Signaling Technology, Danvers, MA), and anti-GS28 (BD Biosciences, San Jose, CA).

Techniques: Plasmid Preparation, Construct, Y2H Assay, Expressing, Western Blot, Activity Assay, Transformation Assay, Transfection, Infection, Pull Down Assay

Journal: Molecular Biology of the Cell

Article Title: p23/Tmp21 Differentially Targets the Rac-GAP β2-Chimaerin and Protein Kinase C via Their C1 Domains

doi: 10.1091/mbc.E09-08-0735

Figure Lengend Snippet: Disruption of β2-chimaerin-p23/Tmp21 interaction leads to enhanced β2-chimaerin Rac-GAP activity. (A) COS-1 cells were transfected with pEGFP, pEGFP-β2-chimaerin (wt), or pEGFP-β2-chimaerin (E227G/L248A). Forty-eight hours later, Rac-GTP levels were assayed using a GST-PBD pull-down assay. (B) Densitometric analysis of Rac-GTP levels relative to control (GFP alone). Data are expressed as mean ± SE of five independent experiments. *p < 0.05 between GFP versus GFP-β2-chim (wt); **p < 0.01 between GFP-β2-chim (wt) versus GFP-β2-chim (E227D/L248A). #p < 0.01 between GFP-β2-chim (wt) (−PMA) versus GFP-β2-chim(wt) (+PMA). (C) Western blots show GFP, GFP-β2-chim (wt) and GFP-β2-chim (E227D/L248A) protein expression.

Article Snippet: The following primary antibodies were used: anti-pLexA (Santa Cruz Biotechnology, Santa Cruz, CA), anti-β-actin (Sigma-Aldrich), anti-V5 (Invitrogen), anti-p23/Tmp21 (ProSci, Poway, CA), anti-GST, anti-hemagglutinin (HA), anti-green fluorescent protein (GFP) (Covance, Emeryville, CA), anti-Rac1 (Millipore, Billerica, MA), anti-PKCε (Cell Signaling Technology, Danvers, MA), and anti-GS28 (BD Biosciences, San Jose, CA).

Techniques: Disruption, Activity Assay, Transfection, Pull Down Assay, Control, Western Blot, Expressing

Journal: OncoTargets and Therapy

Article Title: Exploring Protein Expression Profiles in Lung Cancer Insufficient Microwave Ablation: Implications for Recurrence

doi: 10.2147/OTT.S508577

Figure Lengend Snippet: Differential Expression Proteins

Article Snippet: The primary antibodies used in our study were listed below CTP synthase 1 (CTPS1) (Boster #A06374-2, 1:200), Thymidylate synthetase (TYMS) (Boster #BM5360, 1:100), NME nucleoside diphosphate kinase 3 (NME3) (Proteintech #15136-1-AP, 1:200), Renin binding protein (RENBP) (Bioss #bs-8497R, 1:200), Fucose kinase (FCSK) (Sangon #D127113, 1:300), Pyridoxal phosphatase (PDXP) (Abclonal #A17455, 1:300), and Sulfite oxidase (SUOX) (Proteintech #15075-1-AP, 1:200).

Techniques: Quantitative Proteomics, Binding Assay

Journal: OncoTargets and Therapy

Article Title: Exploring Protein Expression Profiles in Lung Cancer Insufficient Microwave Ablation: Implications for Recurrence

doi: 10.2147/OTT.S508577

Figure Lengend Snippet: Relationship Between DEPs and Prognosis From TCGA Dataset

Article Snippet: The primary antibodies used in our study were listed below CTP synthase 1 (CTPS1) (Boster #A06374-2, 1:200), Thymidylate synthetase (TYMS) (Boster #BM5360, 1:100), NME nucleoside diphosphate kinase 3 (NME3) (Proteintech #15136-1-AP, 1:200), Renin binding protein (RENBP) (Bioss #bs-8497R, 1:200), Fucose kinase (FCSK) (Sangon #D127113, 1:300), Pyridoxal phosphatase (PDXP) (Abclonal #A17455, 1:300), and Sulfite oxidase (SUOX) (Proteintech #15075-1-AP, 1:200).

Techniques:

Journal: OncoTargets and Therapy

Article Title: Exploring Protein Expression Profiles in Lung Cancer Insufficient Microwave Ablation: Implications for Recurrence

doi: 10.2147/OTT.S508577

Figure Lengend Snippet: Differential expression proteins of nucleotide metabolism for predicting survival outcomes. The Kaplan-Meier survival analysis and COX regression analysis were conducted on the CTPS1 ( A ) and TYMS ( B ) using data from the TCGA dataset. ( C ) The distribution of risk scores, survival analysis, and heatmap visualization for patients with a double-gene signature. Upper left: The scatter plot represented the risk score from low to high. Different colors represented different groups. Upper right: Kaplan-Meier survival analysis of the risk model from dataset, comparison among different groups was made by Log rank test. Lower left: the scatter plot distribution represented the risk score of different samples corresponding to the survival time and survival status. The bottom heatmap was the gene expression from the signature. Lower right: the ROC curve of the genes.

Article Snippet: The primary antibodies used in our study were listed below CTP synthase 1 (CTPS1) (Boster #A06374-2, 1:200), Thymidylate synthetase (TYMS) (Boster #BM5360, 1:100), NME nucleoside diphosphate kinase 3 (NME3) (Proteintech #15136-1-AP, 1:200), Renin binding protein (RENBP) (Bioss #bs-8497R, 1:200), Fucose kinase (FCSK) (Sangon #D127113, 1:300), Pyridoxal phosphatase (PDXP) (Abclonal #A17455, 1:300), and Sulfite oxidase (SUOX) (Proteintech #15075-1-AP, 1:200).

Techniques: Quantitative Proteomics, Comparison, Gene Expression