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    ProSci Incorporated anti p23 tmp21
    Differential interaction of C1 domains with <t>p23/Tmp21.</t> (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.
    Anti P23 Tmp21, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+p23+tmp21/pmc02854097-45-17-18?v=ProSci+Incorporated
    Average 90 stars, based on 1 article reviews
    anti p23 tmp21 - by Bioz Stars, 2026-07
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    1) Product Images from "p23/Tmp21 Differentially Targets the Rac-GAP β2-Chimaerin and Protein Kinase C via Their C1 Domains"

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

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E09-08-0735

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

    Techniques Used: Plasmid Preparation, Staining, Confocal Microscopy, Generated

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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

    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).
    Figure Legend 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).

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

    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.
    Figure Legend 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.

    Techniques Used: Confocal Microscopy, Fluorescence, Generated

    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.
    Figure Legend 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.

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

    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.
    Figure Legend 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.

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



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    ProSci Incorporated anti p23 tmp21
    Differential interaction of C1 domains with <t>p23/Tmp21.</t> (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.
    Anti P23 Tmp21, supplied by ProSci Incorporated, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/anti+p23+tmp21/pmc02854097-45-17-18?v=ProSci+Incorporated
    Average 90 stars, based on 1 article reviews
    anti p23 tmp21 - by Bioz Stars, 2026-07
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    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.

    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

    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.

    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

    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.

    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

    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.

    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

    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).

    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

    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.

    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

    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.

    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

    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.

    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