goat anti-rabbit igg Millipore Search Results


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  • 99
    Thermo Fisher goat anti rabbit igg h l cross adsorbed secondary antibody
    Goat Anti Rabbit Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti rabbit igg h l highly cross adsorbed secondary antibody
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    Millipore mouse anti α tubulin
    A PS-flippase at REs contributes to the nuclear localization of YAP. a COS-1 cells at low density or high density were fixed, permeabilized, and stained for YAP and TfnR. Magnified images of the boxed areas around the perinuclear REs are shown in the right column. b COS-1 cells at low density were fixed, permeabilized, and stained for YAP and phosphorylated YAP (S127). A fluorescence intensity profile along the dotted line in the magnified image is shown in the right panel. c qRT-PCR analysis of ATP8A1 mRNA in COS-1 cells treated with ATP8A1 siRNA for 48 h. GAPDH was used as an internal control. d COS-1 cells were treated with control or ATP8A1 siRNA for 48 h. The cells were then fixed, permeabilized, and stained for YAP. e Subcellular localization of YAP in cells in d was examined. f Lysates from cells in d were immunoblotted for the indicated proteins. <t>α-tubulin</t> was used as a loading control. g qRT-PCR analysis of CTGF mRNA from cells in d . GAPDH was used as an internal control. Data are mean ± s.d. from two (for e , n > 40 cells) or three (for c , g ) independent experiments. Statistical significance was determined with two-tailed Student’s t -test (for c , g ) or two-sided Fisher’s exact test (for e ); ** P
    Mouse Anti α Tubulin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5843 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher goat anti mouse igg h l cross adsorbed secondary antibody
    A PS-flippase at REs contributes to the nuclear localization of YAP. a COS-1 cells at low density or high density were fixed, permeabilized, and stained for YAP and TfnR. Magnified images of the boxed areas around the perinuclear REs are shown in the right column. b COS-1 cells at low density were fixed, permeabilized, and stained for YAP and phosphorylated YAP (S127). A fluorescence intensity profile along the dotted line in the magnified image is shown in the right panel. c qRT-PCR analysis of ATP8A1 mRNA in COS-1 cells treated with ATP8A1 siRNA for 48 h. GAPDH was used as an internal control. d COS-1 cells were treated with control or ATP8A1 siRNA for 48 h. The cells were then fixed, permeabilized, and stained for YAP. e Subcellular localization of YAP in cells in d was examined. f Lysates from cells in d were immunoblotted for the indicated proteins. <t>α-tubulin</t> was used as a loading control. g qRT-PCR analysis of CTGF mRNA from cells in d . GAPDH was used as an internal control. Data are mean ± s.d. from two (for e , n > 40 cells) or three (for c , g ) independent experiments. Statistical significance was determined with two-tailed Student’s t -test (for c , g ) or two-sided Fisher’s exact test (for e ); ** P
    Goat Anti Mouse Igg H L Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 14005 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore 3 3 diaminobenzidine
    A PS-flippase at REs contributes to the nuclear localization of YAP. a COS-1 cells at low density or high density were fixed, permeabilized, and stained for YAP and TfnR. Magnified images of the boxed areas around the perinuclear REs are shown in the right column. b COS-1 cells at low density were fixed, permeabilized, and stained for YAP and phosphorylated YAP (S127). A fluorescence intensity profile along the dotted line in the magnified image is shown in the right panel. c qRT-PCR analysis of ATP8A1 mRNA in COS-1 cells treated with ATP8A1 siRNA for 48 h. GAPDH was used as an internal control. d COS-1 cells were treated with control or ATP8A1 siRNA for 48 h. The cells were then fixed, permeabilized, and stained for YAP. e Subcellular localization of YAP in cells in d was examined. f Lysates from cells in d were immunoblotted for the indicated proteins. <t>α-tubulin</t> was used as a loading control. g qRT-PCR analysis of CTGF mRNA from cells in d . GAPDH was used as an internal control. Data are mean ± s.d. from two (for e , n > 40 cells) or three (for c , g ) independent experiments. Statistical significance was determined with two-tailed Student’s t -test (for c , g ) or two-sided Fisher’s exact test (for e ); ** P
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    Thermo Fisher goat anti mouse igg h l highly cross adsorbed secondary antibody
    A PS-flippase at REs contributes to the nuclear localization of YAP. a COS-1 cells at low density or high density were fixed, permeabilized, and stained for YAP and TfnR. Magnified images of the boxed areas around the perinuclear REs are shown in the right column. b COS-1 cells at low density were fixed, permeabilized, and stained for YAP and phosphorylated YAP (S127). A fluorescence intensity profile along the dotted line in the magnified image is shown in the right panel. c qRT-PCR analysis of ATP8A1 mRNA in COS-1 cells treated with ATP8A1 siRNA for 48 h. GAPDH was used as an internal control. d COS-1 cells were treated with control or ATP8A1 siRNA for 48 h. The cells were then fixed, permeabilized, and stained for YAP. e Subcellular localization of YAP in cells in d was examined. f Lysates from cells in d were immunoblotted for the indicated proteins. <t>α-tubulin</t> was used as a loading control. g qRT-PCR analysis of CTGF mRNA from cells in d . GAPDH was used as an internal control. Data are mean ± s.d. from two (for e , n > 40 cells) or three (for c , g ) independent experiments. Statistical significance was determined with two-tailed Student’s t -test (for c , g ) or two-sided Fisher’s exact test (for e ); ** P
    Goat Anti Mouse Igg H L Highly Cross Adsorbed Secondary Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9004 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti β actin
    A PS-flippase at REs contributes to the nuclear localization of YAP. a COS-1 cells at low density or high density were fixed, permeabilized, and stained for YAP and TfnR. Magnified images of the boxed areas around the perinuclear REs are shown in the right column. b COS-1 cells at low density were fixed, permeabilized, and stained for YAP and phosphorylated YAP (S127). A fluorescence intensity profile along the dotted line in the magnified image is shown in the right panel. c qRT-PCR analysis of ATP8A1 mRNA in COS-1 cells treated with ATP8A1 siRNA for 48 h. GAPDH was used as an internal control. d COS-1 cells were treated with control or ATP8A1 siRNA for 48 h. The cells were then fixed, permeabilized, and stained for YAP. e Subcellular localization of YAP in cells in d was examined. f Lysates from cells in d were immunoblotted for the indicated proteins. <t>α-tubulin</t> was used as a loading control. g qRT-PCR analysis of CTGF mRNA from cells in d . GAPDH was used as an internal control. Data are mean ± s.d. from two (for e , n > 40 cells) or three (for c , g ) independent experiments. Statistical significance was determined with two-tailed Student’s t -test (for c , g ) or two-sided Fisher’s exact test (for e ); ** P
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    Image Search Results


    A PS-flippase at REs contributes to the nuclear localization of YAP. a COS-1 cells at low density or high density were fixed, permeabilized, and stained for YAP and TfnR. Magnified images of the boxed areas around the perinuclear REs are shown in the right column. b COS-1 cells at low density were fixed, permeabilized, and stained for YAP and phosphorylated YAP (S127). A fluorescence intensity profile along the dotted line in the magnified image is shown in the right panel. c qRT-PCR analysis of ATP8A1 mRNA in COS-1 cells treated with ATP8A1 siRNA for 48 h. GAPDH was used as an internal control. d COS-1 cells were treated with control or ATP8A1 siRNA for 48 h. The cells were then fixed, permeabilized, and stained for YAP. e Subcellular localization of YAP in cells in d was examined. f Lysates from cells in d were immunoblotted for the indicated proteins. α-tubulin was used as a loading control. g qRT-PCR analysis of CTGF mRNA from cells in d . GAPDH was used as an internal control. Data are mean ± s.d. from two (for e , n > 40 cells) or three (for c , g ) independent experiments. Statistical significance was determined with two-tailed Student’s t -test (for c , g ) or two-sided Fisher’s exact test (for e ); ** P

    Journal: Nature Communications

    Article Title: Endosomal phosphatidylserine is critical for the YAP signalling pathway in proliferating cells

    doi: 10.1038/s41467-017-01255-3

    Figure Lengend Snippet: A PS-flippase at REs contributes to the nuclear localization of YAP. a COS-1 cells at low density or high density were fixed, permeabilized, and stained for YAP and TfnR. Magnified images of the boxed areas around the perinuclear REs are shown in the right column. b COS-1 cells at low density were fixed, permeabilized, and stained for YAP and phosphorylated YAP (S127). A fluorescence intensity profile along the dotted line in the magnified image is shown in the right panel. c qRT-PCR analysis of ATP8A1 mRNA in COS-1 cells treated with ATP8A1 siRNA for 48 h. GAPDH was used as an internal control. d COS-1 cells were treated with control or ATP8A1 siRNA for 48 h. The cells were then fixed, permeabilized, and stained for YAP. e Subcellular localization of YAP in cells in d was examined. f Lysates from cells in d were immunoblotted for the indicated proteins. α-tubulin was used as a loading control. g qRT-PCR analysis of CTGF mRNA from cells in d . GAPDH was used as an internal control. Data are mean ± s.d. from two (for e , n > 40 cells) or three (for c , g ) independent experiments. Statistical significance was determined with two-tailed Student’s t -test (for c , g ) or two-sided Fisher’s exact test (for e ); ** P

    Article Snippet: Antibodies Antibodies used in this study were as follows: rabbit anti-WWP1 (ab43791, dilution 1:1000), mouse anti-β-actin (ab8226, dilution 1:1000), and rabbit anti-EHD1 (ab109747, dilution 1:1000) (Abcam); mouse anti-α-tubulin (T-9026, dilution 1:4000) and mouse anti-Myc (9E10, dilution 1:500) (Sigma); rabbit anti-p-YAP (S127) (#13008, dilution 1:1000 for western blot, #4911, dilution 1:250 for immunofluorescence), rabbit anti-p-Lats1 (S909) (#9157, dilution 1:1000), and rabbit anti-FLAG (#2368, dilution 1:1000 for immunofluorescence and western blotting) (Cell Signaling Technology); mouse anti-TfnR (H68.4, dilution 1:1000 for immunofluorescence and western blotting) and rabbit anti-Rab11 (71-5300, dilution 1:1000 for immunofluorescence and western blotting) (Thermo); rabbit anti-syntaxin 5 (110053, dilution 1:1000, Synaptic Systems); sheep anti-TGN46 (AHP500G, dilution 1:4000, Serotec); goat anti-VPS26A (EB06256, dilution 1:200, Everest Biotech); mouse anti-Lamp2 (sc-18822, dilution 1:200) and rabbit anti-WWP2 (sc-30052, dilution 1:500) (Santa Cruz); rabbit anti-Myc (#06-549, dilution 1:1000, Millipore); rabbit anti-Lats1 (A300-477A, dilution 1:4000, Bethyl Laboratories); sheep anti-GM130 (AF8199, dilution 1:1000, R & D Systems); mouse anti-calnexin (610523, dilution 1:1000 for immunofluorescence and western blotting), mouse anti-EEA1 (610456, dilution 1:1000), mouse anti-GS28 antibody (611184, dilution 1:1000), mouse anti-GM130 (610823, dilution 1:1000), and mouse anti-Itch (611198, dilution 1:1000) (BD Transduction Laboratories); anti-mouse CD63 (RFAC4, dilution 1:1000, Cymbus Biotechnology LTD); mouse anti-KDEL (ADI-SPA-827, dilution 1:1000, Enzo Life Sciences); mouse anti-Ubiquitin (sc-8017, dilution 1:1000, Santa Cruz); sheep anti-mouse-IgG conjugated to horseradish peroxidase (HRP) and donkey anti-rabbit-IgG conjugated to HRP (dilution 1:2000, GE Healthcare); goat anti-rat-IgG conjugated to HRP (dilution 1:2000, American Qalex Antibodies); PierceTM High Sensitivity Streptavidin-HRP (dilution 1:4000, Thermo); and Alexa-Fluor-conjugated secondary antibodies (dilution 1:2000, Invitrogen).

    Techniques: Staining, Fluorescence, Quantitative RT-PCR, Two Tailed Test

    Evectin-2 contributes to the nuclear localization of YAP. a COS-1 cells were treated with control siRNA, evectin-2 siRNA#1, or evectin-2 siRNA#2 for 48 h. The cells were then fixed, permeabilized, and stained for YAP. b Subcellular localization of YAP in cells in a was examined. c qRT-PCR analysis of CTGF mRNA from cells in a . GAPDH was used as an internal control. d COS-1 cells were treated with control, Rab11, or VAMP3 siRNA for 48 h. The cells were then fixed, permeabilized, and stained for YAP. e Subcellular localization of YAP in cells in d was examined. f qRT-PCR analysis of CTGF mRNA in MDA-MB-231 cells depleted of evectin-2. GAPDH was used as an internal control. g MDA-MB-231 cells were treated with evectin-2 siRNA for 72 h. Cells were lysed and immunoblotted for the indicated proteins. α-tubulin was used as a loading control. h MDA-MB-231 cells were treated with siRNA for evectin-2, ATP8A1, or YAP/TAZ for 48 h and then trypsinized. A total of 5 × 10 4 cells were replated and further treated with the same siRNA 24 h after replating. The cells were counted every 24 h after replating. i MDA-MB-231 cells were treated with siRNA for evectin-2, evectin-2/Lats1, or evectin-2/Lats1/Lats2 for 48 h and then trypsinized. A total of 5 × 10 4 cells were replated and further treated with the same siRNA 24 h after replating. The cells were counted 96 h after replating. Data are mean ± s.d. from two (for b , e , n > 40 cells) or three (for c , f , h , i ) independent experiments. Statistical significance was determined with two-tailed Student’s t test (for f ), one-way analysis of variance followed by Tukey–Kramer post hoc test (for c , h , i ), or Kruskal–Wallis test followed by Steel–Dwass post hoc test (for b , e ); ** P

    Journal: Nature Communications

    Article Title: Endosomal phosphatidylserine is critical for the YAP signalling pathway in proliferating cells

    doi: 10.1038/s41467-017-01255-3

    Figure Lengend Snippet: Evectin-2 contributes to the nuclear localization of YAP. a COS-1 cells were treated with control siRNA, evectin-2 siRNA#1, or evectin-2 siRNA#2 for 48 h. The cells were then fixed, permeabilized, and stained for YAP. b Subcellular localization of YAP in cells in a was examined. c qRT-PCR analysis of CTGF mRNA from cells in a . GAPDH was used as an internal control. d COS-1 cells were treated with control, Rab11, or VAMP3 siRNA for 48 h. The cells were then fixed, permeabilized, and stained for YAP. e Subcellular localization of YAP in cells in d was examined. f qRT-PCR analysis of CTGF mRNA in MDA-MB-231 cells depleted of evectin-2. GAPDH was used as an internal control. g MDA-MB-231 cells were treated with evectin-2 siRNA for 72 h. Cells were lysed and immunoblotted for the indicated proteins. α-tubulin was used as a loading control. h MDA-MB-231 cells were treated with siRNA for evectin-2, ATP8A1, or YAP/TAZ for 48 h and then trypsinized. A total of 5 × 10 4 cells were replated and further treated with the same siRNA 24 h after replating. The cells were counted every 24 h after replating. i MDA-MB-231 cells were treated with siRNA for evectin-2, evectin-2/Lats1, or evectin-2/Lats1/Lats2 for 48 h and then trypsinized. A total of 5 × 10 4 cells were replated and further treated with the same siRNA 24 h after replating. The cells were counted 96 h after replating. Data are mean ± s.d. from two (for b , e , n > 40 cells) or three (for c , f , h , i ) independent experiments. Statistical significance was determined with two-tailed Student’s t test (for f ), one-way analysis of variance followed by Tukey–Kramer post hoc test (for c , h , i ), or Kruskal–Wallis test followed by Steel–Dwass post hoc test (for b , e ); ** P

    Article Snippet: Antibodies Antibodies used in this study were as follows: rabbit anti-WWP1 (ab43791, dilution 1:1000), mouse anti-β-actin (ab8226, dilution 1:1000), and rabbit anti-EHD1 (ab109747, dilution 1:1000) (Abcam); mouse anti-α-tubulin (T-9026, dilution 1:4000) and mouse anti-Myc (9E10, dilution 1:500) (Sigma); rabbit anti-p-YAP (S127) (#13008, dilution 1:1000 for western blot, #4911, dilution 1:250 for immunofluorescence), rabbit anti-p-Lats1 (S909) (#9157, dilution 1:1000), and rabbit anti-FLAG (#2368, dilution 1:1000 for immunofluorescence and western blotting) (Cell Signaling Technology); mouse anti-TfnR (H68.4, dilution 1:1000 for immunofluorescence and western blotting) and rabbit anti-Rab11 (71-5300, dilution 1:1000 for immunofluorescence and western blotting) (Thermo); rabbit anti-syntaxin 5 (110053, dilution 1:1000, Synaptic Systems); sheep anti-TGN46 (AHP500G, dilution 1:4000, Serotec); goat anti-VPS26A (EB06256, dilution 1:200, Everest Biotech); mouse anti-Lamp2 (sc-18822, dilution 1:200) and rabbit anti-WWP2 (sc-30052, dilution 1:500) (Santa Cruz); rabbit anti-Myc (#06-549, dilution 1:1000, Millipore); rabbit anti-Lats1 (A300-477A, dilution 1:4000, Bethyl Laboratories); sheep anti-GM130 (AF8199, dilution 1:1000, R & D Systems); mouse anti-calnexin (610523, dilution 1:1000 for immunofluorescence and western blotting), mouse anti-EEA1 (610456, dilution 1:1000), mouse anti-GS28 antibody (611184, dilution 1:1000), mouse anti-GM130 (610823, dilution 1:1000), and mouse anti-Itch (611198, dilution 1:1000) (BD Transduction Laboratories); anti-mouse CD63 (RFAC4, dilution 1:1000, Cymbus Biotechnology LTD); mouse anti-KDEL (ADI-SPA-827, dilution 1:1000, Enzo Life Sciences); mouse anti-Ubiquitin (sc-8017, dilution 1:1000, Santa Cruz); sheep anti-mouse-IgG conjugated to horseradish peroxidase (HRP) and donkey anti-rabbit-IgG conjugated to HRP (dilution 1:2000, GE Healthcare); goat anti-rat-IgG conjugated to HRP (dilution 1:2000, American Qalex Antibodies); PierceTM High Sensitivity Streptavidin-HRP (dilution 1:4000, Thermo); and Alexa-Fluor-conjugated secondary antibodies (dilution 1:2000, Invitrogen).

    Techniques: Staining, Quantitative RT-PCR, Multiple Displacement Amplification, Two Tailed Test

    Cellular PS levels contribute to the nuclear localization of YAP. a GFP, GFP-2xPH (evectin-2, WT), or GFP-2xPH (K20E) was expressed in COS-1 cells for 24 h. The cells were then fixed, permeabilized, and stained for YAP and p-YAP. b Subcellular localization of YAP in cells in a was examined. c The fluorescence intensity of p-YAP in a was quantified and normalized to that of GFP-transfected cells. d Transcriptional coactivity of YAP was examined by the TEAD reporter system. GFP, GFP-2xPH, or GFP-2xPH (K20E) was co-expressed with 8xGTIIC TEAD reporter and pRL-TK (as internal control). e PSA-3 cells were cultured for 72 h with three different conditions (FBS + 10 μM Etn, dFBS, or dFBS + 40 μM Etn). qRT-PCR analysis of CTGF mRNA in these cells was then performed. GAPDH was used as an internal control. f Lysates from cells in e were immunoblotted for the indicated proteins. α-tubulin was used as a loading control. g COS-1 cells were cotransfected with GFP-2xPH and Myc-yPSD1ΔN (WT or S463A). After 24 h, the cells were fixed and stained for Myc. h COS-1 cells were transfected with mKate2, Myc-yPSD1ΔN (WT), or Myc-yPSD1ΔN (S463A). After 24 h, the cells were fixed and costained for YAP, p-YAP, and Myc. i Subcellular localization of YAP in cells in h was examined. j The fluorescence intensity of p-YAP in h was quantified and normalized to that of mKate2-transfected cells. Data are mean ± s.d. from two (for b , c , n > 30 cells), three (for e ), three (for i , j , n > 85 cells), or six (for d ) independent experiments. Statistical significance was determined with one-way analysis of variance followed by Tukey–Kramer post hoc test (for c , d , e , j ) or Kruskal–Wallis test followed by Steel–Dwass post hoc test (for b , i ); * P

    Journal: Nature Communications

    Article Title: Endosomal phosphatidylserine is critical for the YAP signalling pathway in proliferating cells

    doi: 10.1038/s41467-017-01255-3

    Figure Lengend Snippet: Cellular PS levels contribute to the nuclear localization of YAP. a GFP, GFP-2xPH (evectin-2, WT), or GFP-2xPH (K20E) was expressed in COS-1 cells for 24 h. The cells were then fixed, permeabilized, and stained for YAP and p-YAP. b Subcellular localization of YAP in cells in a was examined. c The fluorescence intensity of p-YAP in a was quantified and normalized to that of GFP-transfected cells. d Transcriptional coactivity of YAP was examined by the TEAD reporter system. GFP, GFP-2xPH, or GFP-2xPH (K20E) was co-expressed with 8xGTIIC TEAD reporter and pRL-TK (as internal control). e PSA-3 cells were cultured for 72 h with three different conditions (FBS + 10 μM Etn, dFBS, or dFBS + 40 μM Etn). qRT-PCR analysis of CTGF mRNA in these cells was then performed. GAPDH was used as an internal control. f Lysates from cells in e were immunoblotted for the indicated proteins. α-tubulin was used as a loading control. g COS-1 cells were cotransfected with GFP-2xPH and Myc-yPSD1ΔN (WT or S463A). After 24 h, the cells were fixed and stained for Myc. h COS-1 cells were transfected with mKate2, Myc-yPSD1ΔN (WT), or Myc-yPSD1ΔN (S463A). After 24 h, the cells were fixed and costained for YAP, p-YAP, and Myc. i Subcellular localization of YAP in cells in h was examined. j The fluorescence intensity of p-YAP in h was quantified and normalized to that of mKate2-transfected cells. Data are mean ± s.d. from two (for b , c , n > 30 cells), three (for e ), three (for i , j , n > 85 cells), or six (for d ) independent experiments. Statistical significance was determined with one-way analysis of variance followed by Tukey–Kramer post hoc test (for c , d , e , j ) or Kruskal–Wallis test followed by Steel–Dwass post hoc test (for b , i ); * P

    Article Snippet: Antibodies Antibodies used in this study were as follows: rabbit anti-WWP1 (ab43791, dilution 1:1000), mouse anti-β-actin (ab8226, dilution 1:1000), and rabbit anti-EHD1 (ab109747, dilution 1:1000) (Abcam); mouse anti-α-tubulin (T-9026, dilution 1:4000) and mouse anti-Myc (9E10, dilution 1:500) (Sigma); rabbit anti-p-YAP (S127) (#13008, dilution 1:1000 for western blot, #4911, dilution 1:250 for immunofluorescence), rabbit anti-p-Lats1 (S909) (#9157, dilution 1:1000), and rabbit anti-FLAG (#2368, dilution 1:1000 for immunofluorescence and western blotting) (Cell Signaling Technology); mouse anti-TfnR (H68.4, dilution 1:1000 for immunofluorescence and western blotting) and rabbit anti-Rab11 (71-5300, dilution 1:1000 for immunofluorescence and western blotting) (Thermo); rabbit anti-syntaxin 5 (110053, dilution 1:1000, Synaptic Systems); sheep anti-TGN46 (AHP500G, dilution 1:4000, Serotec); goat anti-VPS26A (EB06256, dilution 1:200, Everest Biotech); mouse anti-Lamp2 (sc-18822, dilution 1:200) and rabbit anti-WWP2 (sc-30052, dilution 1:500) (Santa Cruz); rabbit anti-Myc (#06-549, dilution 1:1000, Millipore); rabbit anti-Lats1 (A300-477A, dilution 1:4000, Bethyl Laboratories); sheep anti-GM130 (AF8199, dilution 1:1000, R & D Systems); mouse anti-calnexin (610523, dilution 1:1000 for immunofluorescence and western blotting), mouse anti-EEA1 (610456, dilution 1:1000), mouse anti-GS28 antibody (611184, dilution 1:1000), mouse anti-GM130 (610823, dilution 1:1000), and mouse anti-Itch (611198, dilution 1:1000) (BD Transduction Laboratories); anti-mouse CD63 (RFAC4, dilution 1:1000, Cymbus Biotechnology LTD); mouse anti-KDEL (ADI-SPA-827, dilution 1:1000, Enzo Life Sciences); mouse anti-Ubiquitin (sc-8017, dilution 1:1000, Santa Cruz); sheep anti-mouse-IgG conjugated to horseradish peroxidase (HRP) and donkey anti-rabbit-IgG conjugated to HRP (dilution 1:2000, GE Healthcare); goat anti-rat-IgG conjugated to HRP (dilution 1:2000, American Qalex Antibodies); PierceTM High Sensitivity Streptavidin-HRP (dilution 1:4000, Thermo); and Alexa-Fluor-conjugated secondary antibodies (dilution 1:2000, Invitrogen).

    Techniques: Staining, Fluorescence, Transfection, Cell Culture, Quantitative RT-PCR

    Binding of evectin-2 and Nedd4 E3 ligases is required for the nuclear localization of YAP. a FLAG-tagged evectin-2 was expressed in COS-1 cells for 24 h. Cell lysates with 1% Triton X-100 were immunoprecipitated with anti-FLAG antibody. The lysate (0.8%) and the immunoprecipitated fraction (10%) were then blotted for FLAG, Itch, WWP1, or WWP2. β-actin was used as a loading control. b Indicated proteins were expressed in COS-1 cells. The cells were then fixed, permeabilized, and stained for Myc. c COS-1 cells were treated with control, Itch, WWP1, or WWP2 siRNA for 48 h. The cells were then fixed, permeabilized, and stained for YAP. d Subcellular localization of YAP in cells in c was examined. e COS-1 cells were treated with the indicated siRNAs for 48 h. qRT-PCR analysis of CTGF mRNA was performed. GAPDH was used as an internal control. f The lysate of COS-1 cells in e was immunoblotted for the indicated proteins. α-tubulin was used as a loading control. A closed arrowhead indicates WWP1. g FLAG-tagged evectin-2 protein (WT or mutants) was expressed in COS-1 cells for 24 h. Cell lysates with 1% Triton X-100 were immunoprecipitated with anti-FLAG antibody. The lysate (0.8%) and the immunoprecipitated fraction (30%) were then blotted for FLAG, Itch, WWP1, or WWP2. α-tubulin was used as a loading control. h COS-1 cells that express siRNA-resistant Myc-evectin-2 WT or PPPA mutant in a cumate-dependent fashion were established. These cells were first treated with control or evectin-2 siRNA for 24 h, and incubated with 30 μM cumate for another 24 h. The cells were then fixed, permeabilized, and stained for YAP and Myc. Arrows indicate cells that express Myc-evt-2 WT or PPPA. i Subcellular localization of YAP in cells in h was examined. j FLAG-WWP2 and Myc-evectin-2 were expressed in HEK293T cells for 24 h. Cell lysates with 1% Triton X-100 were immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were then blotted for ubiquitin (Ub), FLAG, or Myc. α-tubulin was used as a loading control. k A model of the regulation of YAP on RE membranes in proliferating cells. Data are mean ± s.d. from two (for d , n > 40 cells; for i , n > 30 cells) or three (for e ) independent experiments. Statistical significance was determined with one-way analysis of variance followed by Tukey–Kramer post hoc test (for e ) or Kruskal–Wallis test followed by Steel–Dwass post hoc test (for d , i ); ** P

    Journal: Nature Communications

    Article Title: Endosomal phosphatidylserine is critical for the YAP signalling pathway in proliferating cells

    doi: 10.1038/s41467-017-01255-3

    Figure Lengend Snippet: Binding of evectin-2 and Nedd4 E3 ligases is required for the nuclear localization of YAP. a FLAG-tagged evectin-2 was expressed in COS-1 cells for 24 h. Cell lysates with 1% Triton X-100 were immunoprecipitated with anti-FLAG antibody. The lysate (0.8%) and the immunoprecipitated fraction (10%) were then blotted for FLAG, Itch, WWP1, or WWP2. β-actin was used as a loading control. b Indicated proteins were expressed in COS-1 cells. The cells were then fixed, permeabilized, and stained for Myc. c COS-1 cells were treated with control, Itch, WWP1, or WWP2 siRNA for 48 h. The cells were then fixed, permeabilized, and stained for YAP. d Subcellular localization of YAP in cells in c was examined. e COS-1 cells were treated with the indicated siRNAs for 48 h. qRT-PCR analysis of CTGF mRNA was performed. GAPDH was used as an internal control. f The lysate of COS-1 cells in e was immunoblotted for the indicated proteins. α-tubulin was used as a loading control. A closed arrowhead indicates WWP1. g FLAG-tagged evectin-2 protein (WT or mutants) was expressed in COS-1 cells for 24 h. Cell lysates with 1% Triton X-100 were immunoprecipitated with anti-FLAG antibody. The lysate (0.8%) and the immunoprecipitated fraction (30%) were then blotted for FLAG, Itch, WWP1, or WWP2. α-tubulin was used as a loading control. h COS-1 cells that express siRNA-resistant Myc-evectin-2 WT or PPPA mutant in a cumate-dependent fashion were established. These cells were first treated with control or evectin-2 siRNA for 24 h, and incubated with 30 μM cumate for another 24 h. The cells were then fixed, permeabilized, and stained for YAP and Myc. Arrows indicate cells that express Myc-evt-2 WT or PPPA. i Subcellular localization of YAP in cells in h was examined. j FLAG-WWP2 and Myc-evectin-2 were expressed in HEK293T cells for 24 h. Cell lysates with 1% Triton X-100 were immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were then blotted for ubiquitin (Ub), FLAG, or Myc. α-tubulin was used as a loading control. k A model of the regulation of YAP on RE membranes in proliferating cells. Data are mean ± s.d. from two (for d , n > 40 cells; for i , n > 30 cells) or three (for e ) independent experiments. Statistical significance was determined with one-way analysis of variance followed by Tukey–Kramer post hoc test (for e ) or Kruskal–Wallis test followed by Steel–Dwass post hoc test (for d , i ); ** P

    Article Snippet: Antibodies Antibodies used in this study were as follows: rabbit anti-WWP1 (ab43791, dilution 1:1000), mouse anti-β-actin (ab8226, dilution 1:1000), and rabbit anti-EHD1 (ab109747, dilution 1:1000) (Abcam); mouse anti-α-tubulin (T-9026, dilution 1:4000) and mouse anti-Myc (9E10, dilution 1:500) (Sigma); rabbit anti-p-YAP (S127) (#13008, dilution 1:1000 for western blot, #4911, dilution 1:250 for immunofluorescence), rabbit anti-p-Lats1 (S909) (#9157, dilution 1:1000), and rabbit anti-FLAG (#2368, dilution 1:1000 for immunofluorescence and western blotting) (Cell Signaling Technology); mouse anti-TfnR (H68.4, dilution 1:1000 for immunofluorescence and western blotting) and rabbit anti-Rab11 (71-5300, dilution 1:1000 for immunofluorescence and western blotting) (Thermo); rabbit anti-syntaxin 5 (110053, dilution 1:1000, Synaptic Systems); sheep anti-TGN46 (AHP500G, dilution 1:4000, Serotec); goat anti-VPS26A (EB06256, dilution 1:200, Everest Biotech); mouse anti-Lamp2 (sc-18822, dilution 1:200) and rabbit anti-WWP2 (sc-30052, dilution 1:500) (Santa Cruz); rabbit anti-Myc (#06-549, dilution 1:1000, Millipore); rabbit anti-Lats1 (A300-477A, dilution 1:4000, Bethyl Laboratories); sheep anti-GM130 (AF8199, dilution 1:1000, R & D Systems); mouse anti-calnexin (610523, dilution 1:1000 for immunofluorescence and western blotting), mouse anti-EEA1 (610456, dilution 1:1000), mouse anti-GS28 antibody (611184, dilution 1:1000), mouse anti-GM130 (610823, dilution 1:1000), and mouse anti-Itch (611198, dilution 1:1000) (BD Transduction Laboratories); anti-mouse CD63 (RFAC4, dilution 1:1000, Cymbus Biotechnology LTD); mouse anti-KDEL (ADI-SPA-827, dilution 1:1000, Enzo Life Sciences); mouse anti-Ubiquitin (sc-8017, dilution 1:1000, Santa Cruz); sheep anti-mouse-IgG conjugated to horseradish peroxidase (HRP) and donkey anti-rabbit-IgG conjugated to HRP (dilution 1:2000, GE Healthcare); goat anti-rat-IgG conjugated to HRP (dilution 1:2000, American Qalex Antibodies); PierceTM High Sensitivity Streptavidin-HRP (dilution 1:4000, Thermo); and Alexa-Fluor-conjugated secondary antibodies (dilution 1:2000, Invitrogen).

    Techniques: Binding Assay, Immunoprecipitation, Staining, Quantitative RT-PCR, Mutagenesis, Incubation