immunoblotting Search Results


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    GE Healthcare immunoblots
    Immunoblots, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/immunoblots/product/GE Healthcare
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    86
    Cell Signaling Technology Inc immunoblotting
    NPM1 is partially involved in TGF-β-mediated EMT A . A549-Vec and BEAS-Vec cells were treated with NPM1 siRNA (500 nM) prior to administration with TGF-β1 (5 ng/mL) for 24 h. Intracellular expression of NPM1, E-cadherin, N-cadherin, vimentin, phospho-Smad2 and Smad2/3 was detected by <t>immunoblotting.</t> Image intensity was analyzed using ImageJ program ( http://rsbweb.nih.gov./ij/plugins ). B . Cell migration was performed by a wound healing assay using A549-Vec and BEAS-Vec cells co-treated with NPM1 siRNA and TGF-β1. The assay was repeated twice. C . An invasion assay was performed with A549-Vec and BEAS-Vec cells co-treated with NPM1 siRNA and TGF-β1. Scale bar indicates 100 μm. The assay was repeated twice. Each assay was performed in triplicate, and error bars indicate SD. (**; p
    Immunoblotting, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/immunoblotting/product/Cell Signaling Technology Inc
    Average 86 stars, based on 1 article reviews
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    86
    Santa Cruz Biotechnology immunoblotting
    Effects of SIDT2 overexpression on RNA uptake and degradation by lysosomes. (A and B) Outlines of RNA uptake assays (A) and RNA degradation assays (B) using isolated lysosomes. (C) SIDT2 was overexpressed in Neuro2a cells. Protein levels were analyzed by <t>immunoblotting</t> using a goat anti-SIDT2 antibody. (D) The RNA uptake assay I indicated in (A) was performed using 5 μg of total RNA derived from mouse brains and isolated lysosomes derived from cells overexpressing SIDT2, or from control cells transfected with empty vector. Relative RNA levels in the solution outside lysosomes were quantified, and levels of RNA uptake were measured by subtracting RNA levels remaining in solution outside lysosomes from RNA input levels. Mean values are shown with SEM ( n = 3). ***, P
    Immunoblotting, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/immunoblotting/product/Santa Cruz Biotechnology
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    99
    Bio-Rad immunoblotting
    Effects of SIDT2 overexpression on RNA uptake and degradation by lysosomes. (A and B) Outlines of RNA uptake assays (A) and RNA degradation assays (B) using isolated lysosomes. (C) SIDT2 was overexpressed in Neuro2a cells. Protein levels were analyzed by <t>immunoblotting</t> using a goat anti-SIDT2 antibody. (D) The RNA uptake assay I indicated in (A) was performed using 5 μg of total RNA derived from mouse brains and isolated lysosomes derived from cells overexpressing SIDT2, or from control cells transfected with empty vector. Relative RNA levels in the solution outside lysosomes were quantified, and levels of RNA uptake were measured by subtracting RNA levels remaining in solution outside lysosomes from RNA input levels. Mean values are shown with SEM ( n = 3). ***, P
    Immunoblotting, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    N/A
    Protein methionine sulfoxide MetO is a reversible oxidative modification that occurs by exposure of protein s methionine residues to reactive oxygen species ROS Methionine oxidation can alter the function s
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    Image Search Results


    NPM1 is partially involved in TGF-β-mediated EMT A . A549-Vec and BEAS-Vec cells were treated with NPM1 siRNA (500 nM) prior to administration with TGF-β1 (5 ng/mL) for 24 h. Intracellular expression of NPM1, E-cadherin, N-cadherin, vimentin, phospho-Smad2 and Smad2/3 was detected by immunoblotting. Image intensity was analyzed using ImageJ program ( http://rsbweb.nih.gov./ij/plugins ). B . Cell migration was performed by a wound healing assay using A549-Vec and BEAS-Vec cells co-treated with NPM1 siRNA and TGF-β1. The assay was repeated twice. C . An invasion assay was performed with A549-Vec and BEAS-Vec cells co-treated with NPM1 siRNA and TGF-β1. Scale bar indicates 100 μm. The assay was repeated twice. Each assay was performed in triplicate, and error bars indicate SD. (**; p

    Journal: Oncotarget

    Article Title: Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-β signaling

    doi: 10.18632/oncotarget.13867

    Figure Lengend Snippet: NPM1 is partially involved in TGF-β-mediated EMT A . A549-Vec and BEAS-Vec cells were treated with NPM1 siRNA (500 nM) prior to administration with TGF-β1 (5 ng/mL) for 24 h. Intracellular expression of NPM1, E-cadherin, N-cadherin, vimentin, phospho-Smad2 and Smad2/3 was detected by immunoblotting. Image intensity was analyzed using ImageJ program ( http://rsbweb.nih.gov./ij/plugins ). B . Cell migration was performed by a wound healing assay using A549-Vec and BEAS-Vec cells co-treated with NPM1 siRNA and TGF-β1. The assay was repeated twice. C . An invasion assay was performed with A549-Vec and BEAS-Vec cells co-treated with NPM1 siRNA and TGF-β1. Scale bar indicates 100 μm. The assay was repeated twice. Each assay was performed in triplicate, and error bars indicate SD. (**; p

    Article Snippet: Reagents and antibodies For immunoblotting, anti-AKT, -ERK, -JNK, -p38 MAPK, -Smad2/3 antibodies and their corresponding phospho-specific antibodies were acquired from Cell Signaling Biotechnology (Danvers, MA).

    Techniques: Expressing, Migration, Wound Healing Assay, Invasion Assay

    NPM1 silence inhibits TGF-β signaling A . At 48 h post-treatment with NPM1 siRNA (500 nM), A549-CUG2 and BEAS-CUG2 cells were fractionated into cytosolic and nuclear extracts. The whole cell lysates were also prepared at 48 h post-treatment with NPM1 siRNA. Expression of phospho-Smad2, Smad2/3, Snail and Twist was detected by immunoblotting. Sp1 and actin were used loading controls for nuclear and cytosolic extracts, respectively Image intensity was analyzed using ImageJ program ( http://rsbweb.nih.gov./ij/plugins ). B . A549-CUG2 and BEAS-CUG2 cells were incubated on chamber slide followed by fixation and permeabilization at 48 h post-treatment with NPM1 siRNA (500 nM). Expression of Smad2/3 or Snail was detected by immunofluorescence using Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). For nuclear staining, DAPI was added prior to mounting in glycerol. Scale bar indicates 10 μm.

    Journal: Oncotarget

    Article Title: Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-β signaling

    doi: 10.18632/oncotarget.13867

    Figure Lengend Snippet: NPM1 silence inhibits TGF-β signaling A . At 48 h post-treatment with NPM1 siRNA (500 nM), A549-CUG2 and BEAS-CUG2 cells were fractionated into cytosolic and nuclear extracts. The whole cell lysates were also prepared at 48 h post-treatment with NPM1 siRNA. Expression of phospho-Smad2, Smad2/3, Snail and Twist was detected by immunoblotting. Sp1 and actin were used loading controls for nuclear and cytosolic extracts, respectively Image intensity was analyzed using ImageJ program ( http://rsbweb.nih.gov./ij/plugins ). B . A549-CUG2 and BEAS-CUG2 cells were incubated on chamber slide followed by fixation and permeabilization at 48 h post-treatment with NPM1 siRNA (500 nM). Expression of Smad2/3 or Snail was detected by immunofluorescence using Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). For nuclear staining, DAPI was added prior to mounting in glycerol. Scale bar indicates 10 μm.

    Article Snippet: Reagents and antibodies For immunoblotting, anti-AKT, -ERK, -JNK, -p38 MAPK, -Smad2/3 antibodies and their corresponding phospho-specific antibodies were acquired from Cell Signaling Biotechnology (Danvers, MA).

    Techniques: Expressing, Incubation, Immunofluorescence, Staining

    Treatment with Akt or MAPK inhibitors diminishes the CUG2-mediated TGF-β signaling After A549-CUG2 and BEAS-CUG2 cells were treated with wortmannin (Wort; 10 μM), PD98059 (PD; 30 μM), SP600125 (SP; 20 μM), or SB203580 (SB; 30 μM) for 24 h, inactivation of Akt, ERK, JNK and p38 MAPK was confirmed by immunoblotting using their corresponding phospho-specific antibodies. Expression of phospho-Smad2, Smad2/3, Snail and Twist was detected by immunoblotting after cellular fractionations. Sp1 and actin were used loading controls for nuclear and cytosolic extracts, respectively.

    Journal: Oncotarget

    Article Title: Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-β signaling

    doi: 10.18632/oncotarget.13867

    Figure Lengend Snippet: Treatment with Akt or MAPK inhibitors diminishes the CUG2-mediated TGF-β signaling After A549-CUG2 and BEAS-CUG2 cells were treated with wortmannin (Wort; 10 μM), PD98059 (PD; 30 μM), SP600125 (SP; 20 μM), or SB203580 (SB; 30 μM) for 24 h, inactivation of Akt, ERK, JNK and p38 MAPK was confirmed by immunoblotting using their corresponding phospho-specific antibodies. Expression of phospho-Smad2, Smad2/3, Snail and Twist was detected by immunoblotting after cellular fractionations. Sp1 and actin were used loading controls for nuclear and cytosolic extracts, respectively.

    Article Snippet: Reagents and antibodies For immunoblotting, anti-AKT, -ERK, -JNK, -p38 MAPK, -Smad2/3 antibodies and their corresponding phospho-specific antibodies were acquired from Cell Signaling Biotechnology (Danvers, MA).

    Techniques: Expressing

    CUG2 activates Akt and MAPKs, which are dependent on TGF-β signaling, except p38 MAPK A . Activation of Akt, ERK, JNK and p38 MAPK in A549-CUG2 and BEAS-CUG2 cells was compared with those in their control cells by immunoblotting using their corresponding phospho-specific antibodies. B . A549-CUG2 and BEAS-CUG2 cells were treated with EW-7197 at the dose (1.25 μM) for 24 h. Activation of Akt, ERK, JNK and p38 MAPK was examined by immunoblotting.

    Journal: Oncotarget

    Article Title: Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-β signaling

    doi: 10.18632/oncotarget.13867

    Figure Lengend Snippet: CUG2 activates Akt and MAPKs, which are dependent on TGF-β signaling, except p38 MAPK A . Activation of Akt, ERK, JNK and p38 MAPK in A549-CUG2 and BEAS-CUG2 cells was compared with those in their control cells by immunoblotting using their corresponding phospho-specific antibodies. B . A549-CUG2 and BEAS-CUG2 cells were treated with EW-7197 at the dose (1.25 μM) for 24 h. Activation of Akt, ERK, JNK and p38 MAPK was examined by immunoblotting.

    Article Snippet: Reagents and antibodies For immunoblotting, anti-AKT, -ERK, -JNK, -p38 MAPK, -Smad2/3 antibodies and their corresponding phospho-specific antibodies were acquired from Cell Signaling Biotechnology (Danvers, MA).

    Techniques: Activation Assay

    CUG2 is partially involved in TGF-β-mediated EMT A . A549-Vec and BEAS-Vec cells were treated with CUG2 siRNA (500 nM) prior to administration of TGF-β1 (5 ng/mL) for 24 h. Intracellular expression of CUG2, E-cadherin, N-cadherin, vimentin, phospho-Smad2 and Smad2/3 was detected by immunoblotting. Image intensity was analyzed using ImageJ program ( http://rsbweb.nih.gov./ij/plugins ). B . Cell migration was performed by a wound healing assay using A549-Vec and BEAS-Vec cells co-treated with CUG2 siRNA and TGF-β1. C . An invasion assay was performed with A549-Vec and BEAS-Vec cells co-treated with CUG2 siRNA and TGF-β1. Scale bar indicates 100 μm. The assay was repeated twice. Each assay was performed in triplicate, and error bars indicate SD. (**; p

    Journal: Oncotarget

    Article Title: Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-β signaling

    doi: 10.18632/oncotarget.13867

    Figure Lengend Snippet: CUG2 is partially involved in TGF-β-mediated EMT A . A549-Vec and BEAS-Vec cells were treated with CUG2 siRNA (500 nM) prior to administration of TGF-β1 (5 ng/mL) for 24 h. Intracellular expression of CUG2, E-cadherin, N-cadherin, vimentin, phospho-Smad2 and Smad2/3 was detected by immunoblotting. Image intensity was analyzed using ImageJ program ( http://rsbweb.nih.gov./ij/plugins ). B . Cell migration was performed by a wound healing assay using A549-Vec and BEAS-Vec cells co-treated with CUG2 siRNA and TGF-β1. C . An invasion assay was performed with A549-Vec and BEAS-Vec cells co-treated with CUG2 siRNA and TGF-β1. Scale bar indicates 100 μm. The assay was repeated twice. Each assay was performed in triplicate, and error bars indicate SD. (**; p

    Article Snippet: Reagents and antibodies For immunoblotting, anti-AKT, -ERK, -JNK, -p38 MAPK, -Smad2/3 antibodies and their corresponding phospho-specific antibodies were acquired from Cell Signaling Biotechnology (Danvers, MA).

    Techniques: Expressing, Migration, Wound Healing Assay, Invasion Assay

    NPM1 silence inhibits the CUG2-induced EMT A . At 48 h post-treatment with NPM1 siRNA (500 nM), expression of NPM1, E-cadherin, N-cadherin, and vimentin in A549-CUG2 and BEAS-CUG2 cells was detected by immunoblotting. (siCon; control siRNA, siNPM1; NPM1 siRNA) B . A549-CUG2 and BEAS-CUG2 cells were incubated on chamber slide followed by fixation and permeabilization at 48 h post-treatment with NPM1 siRNA(500 nM), Expression of E-cadherin and vimentin was detected by immunofluorescence using Alexa Fluor 488-conjugated goat anti-mouse IgG (green) and Alexa Fluor 488-conjugated donkey anti-goat IgG (green), respectively. For nuclear staining, DAPI was added prior to mounting in glycerol. Scale bar indicates 10 μm. C . Cell migration was measured by a wound healing assay in A549- CUG2 and BEAS-CUG2 cells at 48 h post-treatment with NPM1 siRNA. The wound closure areas were monitored by phase-contrast microscopy at a magnification of 100×. The assays were repeated twice. D . An invasion assay was performed with A549-CUG2 and BEAS-CUG2 at 48 h post-treatment with NPM1 siRNA. Scale bar indicates 100 μm. The assays were repeated twice. Each assay was performed in triplicate and error bars indicate SD (***; p

    Journal: Oncotarget

    Article Title: Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-β signaling

    doi: 10.18632/oncotarget.13867

    Figure Lengend Snippet: NPM1 silence inhibits the CUG2-induced EMT A . At 48 h post-treatment with NPM1 siRNA (500 nM), expression of NPM1, E-cadherin, N-cadherin, and vimentin in A549-CUG2 and BEAS-CUG2 cells was detected by immunoblotting. (siCon; control siRNA, siNPM1; NPM1 siRNA) B . A549-CUG2 and BEAS-CUG2 cells were incubated on chamber slide followed by fixation and permeabilization at 48 h post-treatment with NPM1 siRNA(500 nM), Expression of E-cadherin and vimentin was detected by immunofluorescence using Alexa Fluor 488-conjugated goat anti-mouse IgG (green) and Alexa Fluor 488-conjugated donkey anti-goat IgG (green), respectively. For nuclear staining, DAPI was added prior to mounting in glycerol. Scale bar indicates 10 μm. C . Cell migration was measured by a wound healing assay in A549- CUG2 and BEAS-CUG2 cells at 48 h post-treatment with NPM1 siRNA. The wound closure areas were monitored by phase-contrast microscopy at a magnification of 100×. The assays were repeated twice. D . An invasion assay was performed with A549-CUG2 and BEAS-CUG2 at 48 h post-treatment with NPM1 siRNA. Scale bar indicates 100 μm. The assays were repeated twice. Each assay was performed in triplicate and error bars indicate SD (***; p

    Article Snippet: Reagents and antibodies For immunoblotting, anti-AKT, -ERK, -JNK, -p38 MAPK, -Smad2/3 antibodies and their corresponding phospho-specific antibodies were acquired from Cell Signaling Biotechnology (Danvers, MA).

    Techniques: Expressing, Incubation, Immunofluorescence, Staining, Migration, Wound Healing Assay, Microscopy, Invasion Assay

    Overexpression of CUG2 activates TGF-β signaling A . Expression of phospho-Smad2, Smad2/3, Snail and Twist in A549-CUG2 and BEAS-CUG2 cells was compared with those in their control cells by immunoblotting. In addition, the cells were fractionated into cytosolic and nuclear extracts. Expression of the same proteins was detected by immunoblotting. Sp1 and actin were used loading controls for nuclear and cytosolic extracts, respectively. B . A549-Vec, A549-CUG2, BEAS-Vec and BEAS-CUG2 cells were incubated on chamber slide followed by fixation and permeabilization. Expression of Smad2/3 or Snail was detected by immunofluorescence using Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). For nuclear staining, DAPI was added prior to mounting in glycerol. Scale bar indicates 10 μm.

    Journal: Oncotarget

    Article Title: Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-β signaling

    doi: 10.18632/oncotarget.13867

    Figure Lengend Snippet: Overexpression of CUG2 activates TGF-β signaling A . Expression of phospho-Smad2, Smad2/3, Snail and Twist in A549-CUG2 and BEAS-CUG2 cells was compared with those in their control cells by immunoblotting. In addition, the cells were fractionated into cytosolic and nuclear extracts. Expression of the same proteins was detected by immunoblotting. Sp1 and actin were used loading controls for nuclear and cytosolic extracts, respectively. B . A549-Vec, A549-CUG2, BEAS-Vec and BEAS-CUG2 cells were incubated on chamber slide followed by fixation and permeabilization. Expression of Smad2/3 or Snail was detected by immunofluorescence using Alexa Fluor 488-conjugated goat anti-rabbit IgG (green). For nuclear staining, DAPI was added prior to mounting in glycerol. Scale bar indicates 10 μm.

    Article Snippet: Reagents and antibodies For immunoblotting, anti-AKT, -ERK, -JNK, -p38 MAPK, -Smad2/3 antibodies and their corresponding phospho-specific antibodies were acquired from Cell Signaling Biotechnology (Danvers, MA).

    Techniques: Over Expression, Expressing, Incubation, Immunofluorescence, Staining

    CUG2 silence inhibits TGF-β signaling, leading to suppression of the EMT A . At 48 h post-treatment with CUG siRNA (500 nM), expression of CUG2, E-cadherin, N-cadherin, vimentin, phospho-Smad2, Smad2/3, Snail and Twist in A549-CUG2 and BEAS-CUG2 cells was detected by immunoblotting. (siCon; control siRNA, siCUG2; CUG2 siRNA). B . Cell migration was measured by a wound healing assay in A549- CUG2 and BEAS-CUG2 cells at 48 h post-treatment with CUG2 siRNA. The wound closure areas were monitored by phase-contrast microscopy at a magnification of 100×. The assays were repeated twice. C . An invasion assay was performed with A549-CUG2 and BEAS-CUG2 at 48 h post-treatment with NPM1 siRNA. Scale bar indicates 100 μm. The assays were repeated twice. Each assay was performed in triplicate and error bars indicate SD (***; p

    Journal: Oncotarget

    Article Title: Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-β signaling

    doi: 10.18632/oncotarget.13867

    Figure Lengend Snippet: CUG2 silence inhibits TGF-β signaling, leading to suppression of the EMT A . At 48 h post-treatment with CUG siRNA (500 nM), expression of CUG2, E-cadherin, N-cadherin, vimentin, phospho-Smad2, Smad2/3, Snail and Twist in A549-CUG2 and BEAS-CUG2 cells was detected by immunoblotting. (siCon; control siRNA, siCUG2; CUG2 siRNA). B . Cell migration was measured by a wound healing assay in A549- CUG2 and BEAS-CUG2 cells at 48 h post-treatment with CUG2 siRNA. The wound closure areas were monitored by phase-contrast microscopy at a magnification of 100×. The assays were repeated twice. C . An invasion assay was performed with A549-CUG2 and BEAS-CUG2 at 48 h post-treatment with NPM1 siRNA. Scale bar indicates 100 μm. The assays were repeated twice. Each assay was performed in triplicate and error bars indicate SD (***; p

    Article Snippet: Reagents and antibodies For immunoblotting, anti-AKT, -ERK, -JNK, -p38 MAPK, -Smad2/3 antibodies and their corresponding phospho-specific antibodies were acquired from Cell Signaling Biotechnology (Danvers, MA).

    Techniques: Expressing, Migration, Wound Healing Assay, Microscopy, Invasion Assay

    Treatment with EW-7197 inhibits the CUG2-induced EMT A . A549-CUG2 and BEAS-CUG2 cells were treated with EW-7197 at different doses (0.25, 0.5, 1.25 and 2.5 μM) for 24 h. Expression of CUG2, E-cadherin, N-cadherin, and vimentin was detected by immunoblotting. B . A549-CUG2 or BEAS-CUG2 cells were transfected with CUG2 promoter vectors (F961 and F961-94). At 48 h post-transfection, luciferase enzyme activities were measured in the transfected cell lysates. Transfection efficiency was normalized with the β-galactosidase reporter vector, pGK-β-gal. The assays were repeated in triplicate. The results shown are the average of triplicate wells. Error bars indicate SD. (**; p

    Journal: Oncotarget

    Article Title: Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-β signaling

    doi: 10.18632/oncotarget.13867

    Figure Lengend Snippet: Treatment with EW-7197 inhibits the CUG2-induced EMT A . A549-CUG2 and BEAS-CUG2 cells were treated with EW-7197 at different doses (0.25, 0.5, 1.25 and 2.5 μM) for 24 h. Expression of CUG2, E-cadherin, N-cadherin, and vimentin was detected by immunoblotting. B . A549-CUG2 or BEAS-CUG2 cells were transfected with CUG2 promoter vectors (F961 and F961-94). At 48 h post-transfection, luciferase enzyme activities were measured in the transfected cell lysates. Transfection efficiency was normalized with the β-galactosidase reporter vector, pGK-β-gal. The assays were repeated in triplicate. The results shown are the average of triplicate wells. Error bars indicate SD. (**; p

    Article Snippet: Reagents and antibodies For immunoblotting, anti-AKT, -ERK, -JNK, -p38 MAPK, -Smad2/3 antibodies and their corresponding phospho-specific antibodies were acquired from Cell Signaling Biotechnology (Danvers, MA).

    Techniques: Expressing, Transfection, Luciferase, Plasmid Preparation

    CUG2 induces EMT, in which NT of CUG2 is more important than CT of CUG2 A . Expression of CUG2, E-cadherin, N-cadherin, and vimentin was detected by immunoblotting using the corresponding antibodies. NT indicates N-terminal domain of CUG2 and CT indicates C-terminal domain of CUG2. B . Expression of E-cadherin and vimentin was detected by immunofluorescence using Alexa Fluor 488-conjugated goat anti-mouse IgG (green) and Alexa Fluor 488-conjugated donkey anti-goat IgG (green), respectively. For nuclear staining, DAPI was added prior to mounting in glycerol. Scale bar indicates 10 μm. C . Cell migration was measured by a wound healing assay. The wound closure areas were monitored by phase-contrast microscopy at a magnification of 100×. The assays were repeated twice. D . An invasion assay was performed using 48-well Boyden chambers. The chamber was assembled using polycarbonate filters coated with Matrigel. Scale bar indicates 100 μm. The assays were repeated twice. Each assay was performed in triplicate and error bars indicate standard deviation (SD) (ns; not significant, p > 0.05, ***; p

    Journal: Oncotarget

    Article Title: Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-β signaling

    doi: 10.18632/oncotarget.13867

    Figure Lengend Snippet: CUG2 induces EMT, in which NT of CUG2 is more important than CT of CUG2 A . Expression of CUG2, E-cadherin, N-cadherin, and vimentin was detected by immunoblotting using the corresponding antibodies. NT indicates N-terminal domain of CUG2 and CT indicates C-terminal domain of CUG2. B . Expression of E-cadherin and vimentin was detected by immunofluorescence using Alexa Fluor 488-conjugated goat anti-mouse IgG (green) and Alexa Fluor 488-conjugated donkey anti-goat IgG (green), respectively. For nuclear staining, DAPI was added prior to mounting in glycerol. Scale bar indicates 10 μm. C . Cell migration was measured by a wound healing assay. The wound closure areas were monitored by phase-contrast microscopy at a magnification of 100×. The assays were repeated twice. D . An invasion assay was performed using 48-well Boyden chambers. The chamber was assembled using polycarbonate filters coated with Matrigel. Scale bar indicates 100 μm. The assays were repeated twice. Each assay was performed in triplicate and error bars indicate standard deviation (SD) (ns; not significant, p > 0.05, ***; p

    Article Snippet: Reagents and antibodies For immunoblotting, anti-AKT, -ERK, -JNK, -p38 MAPK, -Smad2/3 antibodies and their corresponding phospho-specific antibodies were acquired from Cell Signaling Biotechnology (Danvers, MA).

    Techniques: Expressing, Immunofluorescence, Staining, Migration, Wound Healing Assay, Microscopy, Invasion Assay, Standard Deviation

    Sp1 transcription factor is required for both CUG2 and TGF-β transcription A . Production of TGF-β protein was detected with immunoblotting with an anti-TGF-β antibody after running of SDS-PAGE under a reduced condition. Production of TGF-β1 protein in the culture media from A549-CUG2 and BEAS-CUG2 cells was compared to that from their control cell media with a modified sandwich ELISA using Au nanoparticles. The assay was repeated in triplicate. The results shown are the average of triplicate wells and error bars indicate SD. (*; p

    Journal: Oncotarget

    Article Title: Cancer upregulated gene 2 induces epithelial-mesenchymal transition of human lung cancer cells via TGF-β signaling

    doi: 10.18632/oncotarget.13867

    Figure Lengend Snippet: Sp1 transcription factor is required for both CUG2 and TGF-β transcription A . Production of TGF-β protein was detected with immunoblotting with an anti-TGF-β antibody after running of SDS-PAGE under a reduced condition. Production of TGF-β1 protein in the culture media from A549-CUG2 and BEAS-CUG2 cells was compared to that from their control cell media with a modified sandwich ELISA using Au nanoparticles. The assay was repeated in triplicate. The results shown are the average of triplicate wells and error bars indicate SD. (*; p

    Article Snippet: Reagents and antibodies For immunoblotting, anti-AKT, -ERK, -JNK, -p38 MAPK, -Smad2/3 antibodies and their corresponding phospho-specific antibodies were acquired from Cell Signaling Biotechnology (Danvers, MA).

    Techniques: SDS Page, Modification, Sandwich ELISA

    Effects of SIDT2 overexpression on RNA uptake and degradation by lysosomes. (A and B) Outlines of RNA uptake assays (A) and RNA degradation assays (B) using isolated lysosomes. (C) SIDT2 was overexpressed in Neuro2a cells. Protein levels were analyzed by immunoblotting using a goat anti-SIDT2 antibody. (D) The RNA uptake assay I indicated in (A) was performed using 5 μg of total RNA derived from mouse brains and isolated lysosomes derived from cells overexpressing SIDT2, or from control cells transfected with empty vector. Relative RNA levels in the solution outside lysosomes were quantified, and levels of RNA uptake were measured by subtracting RNA levels remaining in solution outside lysosomes from RNA input levels. Mean values are shown with SEM ( n = 3). ***, P

    Journal: Autophagy

    Article Title: Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes

    doi: 10.1080/15548627.2016.1145325

    Figure Lengend Snippet: Effects of SIDT2 overexpression on RNA uptake and degradation by lysosomes. (A and B) Outlines of RNA uptake assays (A) and RNA degradation assays (B) using isolated lysosomes. (C) SIDT2 was overexpressed in Neuro2a cells. Protein levels were analyzed by immunoblotting using a goat anti-SIDT2 antibody. (D) The RNA uptake assay I indicated in (A) was performed using 5 μg of total RNA derived from mouse brains and isolated lysosomes derived from cells overexpressing SIDT2, or from control cells transfected with empty vector. Relative RNA levels in the solution outside lysosomes were quantified, and levels of RNA uptake were measured by subtracting RNA levels remaining in solution outside lysosomes from RNA input levels. Mean values are shown with SEM ( n = 3). ***, P

    Article Snippet: The primary antibodies used for immunoprecipitation or immunoblotting were: polyclonal goat anti-SIDT2 (N-20; Santa Cruz Biotechnology, sc-54151), polyclonal goat anti-SIDT1 (GeneTex, GTX88799), monoclonal rat anti-LAMP2 (M3/84; Santa Cruz Biotechnology, sc-19991), monoclonal mouse anti-DYKDDDDK (FLAG; Wako Pure Chemical Industries, 018-22783), monoclonal rabbit anti-SCARB2/LIMP2 (EPR12080; Abcam, ab176317), monoclonal mouse anti-RAB7A (Rab7-117; Abcam, ab50533), monoclonal mouse anti-RAB5A (RAB5A, member RAS oncogene family) (D-11; Santa Cruz Biotechnology, sc-46692), monoclonal mouse anti-MAP1LC3A/B (4E12; MBL, M152-3), polyclonal rabbit anti-LMNA (lamin A) (H-102; Santa Cruz Biotechnology, sc-20680), monoclonal mouse anti-COX4I1 (cytochrome c oxidase subunit IV isoform 1) (20E8C12; Life Technologies, A21348), monoclonal mouse anti-GOLGA1/Golgin-97 (CDF4; Life Technologies, A-21270), polyclonal rabbit anti-CANX (calnexin) (Abcam, ab22595), monoclonal mouse anti-GAPDH (6C5; Merck Millipore, CB1001), polyclonal rabbit anti-TUBB (tubulin, β)/β-tubulin (Cell Signaling Technology, 2146), and monoclonal mouse anti-ACTB/β-actin (AC-15; Sigma-Aldrich, A5441).

    Techniques: Over Expression, Isolation, Derivative Assay, Transfection, Plasmid Preparation

    Effect of SIDT1 overexpression on RNautophagy. (A) Neuro2a cells expressing GFP-tagged SIDT1 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified (right panel, n=3). (B) SIDT1 was overexpressed in Neuro2a cells. Protein levels were analyzed by immunoblotting using an anti-SIDT1 antibody. (C) Lysosomes were isolated from Neuro2a cells overexpressing SIDT1 or control transfectants. The RNA uptake assay I indicated in Fig. 2A was performed. Relative levels of RNA uptake were quantified. Mean ± SEM ( n = 3). n.s., not significant.

    Journal: Autophagy

    Article Title: Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes

    doi: 10.1080/15548627.2016.1145325

    Figure Lengend Snippet: Effect of SIDT1 overexpression on RNautophagy. (A) Neuro2a cells expressing GFP-tagged SIDT1 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified (right panel, n=3). (B) SIDT1 was overexpressed in Neuro2a cells. Protein levels were analyzed by immunoblotting using an anti-SIDT1 antibody. (C) Lysosomes were isolated from Neuro2a cells overexpressing SIDT1 or control transfectants. The RNA uptake assay I indicated in Fig. 2A was performed. Relative levels of RNA uptake were quantified. Mean ± SEM ( n = 3). n.s., not significant.

    Article Snippet: The primary antibodies used for immunoprecipitation or immunoblotting were: polyclonal goat anti-SIDT2 (N-20; Santa Cruz Biotechnology, sc-54151), polyclonal goat anti-SIDT1 (GeneTex, GTX88799), monoclonal rat anti-LAMP2 (M3/84; Santa Cruz Biotechnology, sc-19991), monoclonal mouse anti-DYKDDDDK (FLAG; Wako Pure Chemical Industries, 018-22783), monoclonal rabbit anti-SCARB2/LIMP2 (EPR12080; Abcam, ab176317), monoclonal mouse anti-RAB7A (Rab7-117; Abcam, ab50533), monoclonal mouse anti-RAB5A (RAB5A, member RAS oncogene family) (D-11; Santa Cruz Biotechnology, sc-46692), monoclonal mouse anti-MAP1LC3A/B (4E12; MBL, M152-3), polyclonal rabbit anti-LMNA (lamin A) (H-102; Santa Cruz Biotechnology, sc-20680), monoclonal mouse anti-COX4I1 (cytochrome c oxidase subunit IV isoform 1) (20E8C12; Life Technologies, A21348), monoclonal mouse anti-GOLGA1/Golgin-97 (CDF4; Life Technologies, A-21270), polyclonal rabbit anti-CANX (calnexin) (Abcam, ab22595), monoclonal mouse anti-GAPDH (6C5; Merck Millipore, CB1001), polyclonal rabbit anti-TUBB (tubulin, β)/β-tubulin (Cell Signaling Technology, 2146), and monoclonal mouse anti-ACTB/β-actin (AC-15; Sigma-Aldrich, A5441).

    Techniques: Over Expression, Expressing, Incubation, Fluorescence, Laser-Scanning Microscopy, Isolation

    Effect of SIDT2 mutation on RNautophagy. (A) Neuro2a cells expressing GFP-tagged SIDT2 S564A were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified (right panel, n=3). (B and D) Lysosomes were isolated from Neuro2a cells overexpressing WT or mutant SIDT2 S564A or control transfectants. SIDT2 levels in lysosomes were analyzed by immunoblotting (B). The RNA uptake assay I indicated in Fig. 2A was performed (D). Relative levels of RNA uptake were quantified. Mean ± SEM ( n = 3). ***, P

    Journal: Autophagy

    Article Title: Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes

    doi: 10.1080/15548627.2016.1145325

    Figure Lengend Snippet: Effect of SIDT2 mutation on RNautophagy. (A) Neuro2a cells expressing GFP-tagged SIDT2 S564A were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified (right panel, n=3). (B and D) Lysosomes were isolated from Neuro2a cells overexpressing WT or mutant SIDT2 S564A or control transfectants. SIDT2 levels in lysosomes were analyzed by immunoblotting (B). The RNA uptake assay I indicated in Fig. 2A was performed (D). Relative levels of RNA uptake were quantified. Mean ± SEM ( n = 3). ***, P

    Article Snippet: The primary antibodies used for immunoprecipitation or immunoblotting were: polyclonal goat anti-SIDT2 (N-20; Santa Cruz Biotechnology, sc-54151), polyclonal goat anti-SIDT1 (GeneTex, GTX88799), monoclonal rat anti-LAMP2 (M3/84; Santa Cruz Biotechnology, sc-19991), monoclonal mouse anti-DYKDDDDK (FLAG; Wako Pure Chemical Industries, 018-22783), monoclonal rabbit anti-SCARB2/LIMP2 (EPR12080; Abcam, ab176317), monoclonal mouse anti-RAB7A (Rab7-117; Abcam, ab50533), monoclonal mouse anti-RAB5A (RAB5A, member RAS oncogene family) (D-11; Santa Cruz Biotechnology, sc-46692), monoclonal mouse anti-MAP1LC3A/B (4E12; MBL, M152-3), polyclonal rabbit anti-LMNA (lamin A) (H-102; Santa Cruz Biotechnology, sc-20680), monoclonal mouse anti-COX4I1 (cytochrome c oxidase subunit IV isoform 1) (20E8C12; Life Technologies, A21348), monoclonal mouse anti-GOLGA1/Golgin-97 (CDF4; Life Technologies, A-21270), polyclonal rabbit anti-CANX (calnexin) (Abcam, ab22595), monoclonal mouse anti-GAPDH (6C5; Merck Millipore, CB1001), polyclonal rabbit anti-TUBB (tubulin, β)/β-tubulin (Cell Signaling Technology, 2146), and monoclonal mouse anti-ACTB/β-actin (AC-15; Sigma-Aldrich, A5441).

    Techniques: Mutagenesis, Expressing, Incubation, Fluorescence, Laser-Scanning Microscopy, Isolation

    Effects of Sidt2 knockdown on cellular RNA degradation. (A) Experimental paradigm for monitoring the degradation of cellular RNA. CQ, chloroquine. (B, F, H and K) Decreased levels of SIDT2 proteins in atg5 KO MEFs and in WT MEFs transfected with Sidt2 -siRNA were confirmed by immunoblotting. Mean ± SEM ( n = 4). ***, P

    Journal: Autophagy

    Article Title: Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes

    doi: 10.1080/15548627.2016.1145325

    Figure Lengend Snippet: Effects of Sidt2 knockdown on cellular RNA degradation. (A) Experimental paradigm for monitoring the degradation of cellular RNA. CQ, chloroquine. (B, F, H and K) Decreased levels of SIDT2 proteins in atg5 KO MEFs and in WT MEFs transfected with Sidt2 -siRNA were confirmed by immunoblotting. Mean ± SEM ( n = 4). ***, P

    Article Snippet: The primary antibodies used for immunoprecipitation or immunoblotting were: polyclonal goat anti-SIDT2 (N-20; Santa Cruz Biotechnology, sc-54151), polyclonal goat anti-SIDT1 (GeneTex, GTX88799), monoclonal rat anti-LAMP2 (M3/84; Santa Cruz Biotechnology, sc-19991), monoclonal mouse anti-DYKDDDDK (FLAG; Wako Pure Chemical Industries, 018-22783), monoclonal rabbit anti-SCARB2/LIMP2 (EPR12080; Abcam, ab176317), monoclonal mouse anti-RAB7A (Rab7-117; Abcam, ab50533), monoclonal mouse anti-RAB5A (RAB5A, member RAS oncogene family) (D-11; Santa Cruz Biotechnology, sc-46692), monoclonal mouse anti-MAP1LC3A/B (4E12; MBL, M152-3), polyclonal rabbit anti-LMNA (lamin A) (H-102; Santa Cruz Biotechnology, sc-20680), monoclonal mouse anti-COX4I1 (cytochrome c oxidase subunit IV isoform 1) (20E8C12; Life Technologies, A21348), monoclonal mouse anti-GOLGA1/Golgin-97 (CDF4; Life Technologies, A-21270), polyclonal rabbit anti-CANX (calnexin) (Abcam, ab22595), monoclonal mouse anti-GAPDH (6C5; Merck Millipore, CB1001), polyclonal rabbit anti-TUBB (tubulin, β)/β-tubulin (Cell Signaling Technology, 2146), and monoclonal mouse anti-ACTB/β-actin (AC-15; Sigma-Aldrich, A5441).

    Techniques: Transfection

    Characterization of SIDT2. (A) Lysosomes (Lys) were isolated from mouse brain homogenates (Hom), and analyzed by immunoblotting using polyclonal goat anti-SIDT2 antibody and antibodies against LAMP2 (lysosomal marker), RAB7A (late endosome and lysosome), RAB5A (early endosome), CANX (endoplasmic reticulum), COX4I1 (mitochondria), GOLGA1 (Golgi apparatus), GAPDH (cytosol), LMNA/lamin A (nuclei), and MAP1LC3A/B (autophagosome). (B) Neuro2a cells expressing GFP-tagged SIDT2 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified using ImageJ software (right panel, n = 3). ( C ) Neuro2a cells expressing GFP-tagged SIDT2 were fixed, and immunostained using anti-RAB7A, anti-EEA1 (early endosomal marker) or anti-MAP1LC3A/B antibodies. Fluorescent images were obtained using confocal microscopy. Scale bars: 5 μm. Colocalization rate was quantified (right panels, n = 3). (D) Lysosomes were isolated from HeLa cells expressing SIDT2-FLAG or CTSB-FLAG. Isolated lysosomes (4 μg protein) were incubated with the indicated concentrations of trypsin at 37°C for 5 min. Proteins in the samples were analyzed by immunoblotting using an anti-FLAG antibody. (E) LAMP2C and SIDT2 or SIDT2-FLAG were overexpressed in HeLa cells. Cell lysates were prepared and immunoprecipitated with an anti-FLAG antibody. Cell lysates and the resulting immunoprecipitant were analyzed by immunoblotting. (F) Lysates were prepared from HeLa cells overexpressing SIDT2 and LAMP2C or LAMP2C-FLAG and coimmunoprecipitation assays performed. (G) Endogenous interaction of SIDT2 with LAMP2C. Coimmunoprecipitation assays were performed using mouse brain lysates. (H) Neuro2a cells coexpressing FLAG-tagged LAMP2C and GFP-tagged SIDT2 were fixed, and immunostained using anti-FLAG antibody. Scale bars: 10 μm. Colocalization rate was quantified (right panel, n = 3).

    Journal: Autophagy

    Article Title: Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes

    doi: 10.1080/15548627.2016.1145325

    Figure Lengend Snippet: Characterization of SIDT2. (A) Lysosomes (Lys) were isolated from mouse brain homogenates (Hom), and analyzed by immunoblotting using polyclonal goat anti-SIDT2 antibody and antibodies against LAMP2 (lysosomal marker), RAB7A (late endosome and lysosome), RAB5A (early endosome), CANX (endoplasmic reticulum), COX4I1 (mitochondria), GOLGA1 (Golgi apparatus), GAPDH (cytosol), LMNA/lamin A (nuclei), and MAP1LC3A/B (autophagosome). (B) Neuro2a cells expressing GFP-tagged SIDT2 were incubated with LysoTracker Red. Fluorescence images were visualized using a confocal laser-scanning microscope. Scale bar: 10 μm. Colocalization rate was quantified using ImageJ software (right panel, n = 3). ( C ) Neuro2a cells expressing GFP-tagged SIDT2 were fixed, and immunostained using anti-RAB7A, anti-EEA1 (early endosomal marker) or anti-MAP1LC3A/B antibodies. Fluorescent images were obtained using confocal microscopy. Scale bars: 5 μm. Colocalization rate was quantified (right panels, n = 3). (D) Lysosomes were isolated from HeLa cells expressing SIDT2-FLAG or CTSB-FLAG. Isolated lysosomes (4 μg protein) were incubated with the indicated concentrations of trypsin at 37°C for 5 min. Proteins in the samples were analyzed by immunoblotting using an anti-FLAG antibody. (E) LAMP2C and SIDT2 or SIDT2-FLAG were overexpressed in HeLa cells. Cell lysates were prepared and immunoprecipitated with an anti-FLAG antibody. Cell lysates and the resulting immunoprecipitant were analyzed by immunoblotting. (F) Lysates were prepared from HeLa cells overexpressing SIDT2 and LAMP2C or LAMP2C-FLAG and coimmunoprecipitation assays performed. (G) Endogenous interaction of SIDT2 with LAMP2C. Coimmunoprecipitation assays were performed using mouse brain lysates. (H) Neuro2a cells coexpressing FLAG-tagged LAMP2C and GFP-tagged SIDT2 were fixed, and immunostained using anti-FLAG antibody. Scale bars: 10 μm. Colocalization rate was quantified (right panel, n = 3).

    Article Snippet: The primary antibodies used for immunoprecipitation or immunoblotting were: polyclonal goat anti-SIDT2 (N-20; Santa Cruz Biotechnology, sc-54151), polyclonal goat anti-SIDT1 (GeneTex, GTX88799), monoclonal rat anti-LAMP2 (M3/84; Santa Cruz Biotechnology, sc-19991), monoclonal mouse anti-DYKDDDDK (FLAG; Wako Pure Chemical Industries, 018-22783), monoclonal rabbit anti-SCARB2/LIMP2 (EPR12080; Abcam, ab176317), monoclonal mouse anti-RAB7A (Rab7-117; Abcam, ab50533), monoclonal mouse anti-RAB5A (RAB5A, member RAS oncogene family) (D-11; Santa Cruz Biotechnology, sc-46692), monoclonal mouse anti-MAP1LC3A/B (4E12; MBL, M152-3), polyclonal rabbit anti-LMNA (lamin A) (H-102; Santa Cruz Biotechnology, sc-20680), monoclonal mouse anti-COX4I1 (cytochrome c oxidase subunit IV isoform 1) (20E8C12; Life Technologies, A21348), monoclonal mouse anti-GOLGA1/Golgin-97 (CDF4; Life Technologies, A-21270), polyclonal rabbit anti-CANX (calnexin) (Abcam, ab22595), monoclonal mouse anti-GAPDH (6C5; Merck Millipore, CB1001), polyclonal rabbit anti-TUBB (tubulin, β)/β-tubulin (Cell Signaling Technology, 2146), and monoclonal mouse anti-ACTB/β-actin (AC-15; Sigma-Aldrich, A5441).

    Techniques: Isolation, Marker, Expressing, Incubation, Fluorescence, Laser-Scanning Microscopy, Software, Confocal Microscopy, Immunoprecipitation

    Effect of SIDT2 overexpression on RNautophagy in the absence of LAMP2. (A) LAMP2 levels in LAMP2 -deficient HeLa cells and parental HeLa cells (control HeLa) were analyzed by immunoblotting. (B and C) RNA uptake assays were performed using isolated lysosomes derived from LAMP2 -deficient HeLa cells (B) or parental HeLa cells (C). Relative levels of RNA uptake were quantified. Results are expressed as mean ± SEM ( n = 3). **, P

    Journal: Autophagy

    Article Title: Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes

    doi: 10.1080/15548627.2016.1145325

    Figure Lengend Snippet: Effect of SIDT2 overexpression on RNautophagy in the absence of LAMP2. (A) LAMP2 levels in LAMP2 -deficient HeLa cells and parental HeLa cells (control HeLa) were analyzed by immunoblotting. (B and C) RNA uptake assays were performed using isolated lysosomes derived from LAMP2 -deficient HeLa cells (B) or parental HeLa cells (C). Relative levels of RNA uptake were quantified. Results are expressed as mean ± SEM ( n = 3). **, P

    Article Snippet: The primary antibodies used for immunoprecipitation or immunoblotting were: polyclonal goat anti-SIDT2 (N-20; Santa Cruz Biotechnology, sc-54151), polyclonal goat anti-SIDT1 (GeneTex, GTX88799), monoclonal rat anti-LAMP2 (M3/84; Santa Cruz Biotechnology, sc-19991), monoclonal mouse anti-DYKDDDDK (FLAG; Wako Pure Chemical Industries, 018-22783), monoclonal rabbit anti-SCARB2/LIMP2 (EPR12080; Abcam, ab176317), monoclonal mouse anti-RAB7A (Rab7-117; Abcam, ab50533), monoclonal mouse anti-RAB5A (RAB5A, member RAS oncogene family) (D-11; Santa Cruz Biotechnology, sc-46692), monoclonal mouse anti-MAP1LC3A/B (4E12; MBL, M152-3), polyclonal rabbit anti-LMNA (lamin A) (H-102; Santa Cruz Biotechnology, sc-20680), monoclonal mouse anti-COX4I1 (cytochrome c oxidase subunit IV isoform 1) (20E8C12; Life Technologies, A21348), monoclonal mouse anti-GOLGA1/Golgin-97 (CDF4; Life Technologies, A-21270), polyclonal rabbit anti-CANX (calnexin) (Abcam, ab22595), monoclonal mouse anti-GAPDH (6C5; Merck Millipore, CB1001), polyclonal rabbit anti-TUBB (tubulin, β)/β-tubulin (Cell Signaling Technology, 2146), and monoclonal mouse anti-ACTB/β-actin (AC-15; Sigma-Aldrich, A5441).

    Techniques: Over Expression, Isolation, Derivative Assay

    Effects of SIDT2 knockdown on RNA uptake and degradation by lysosomes. (A and D) Decreased levels of SIDT2 proteins in HeLa cells transfected with SIDT2 -siRNA were confirmed by immunoblotting. Relative levels of SIDT2 were quantified. Results are expressed as mean ± SEM ( n = 3). (B and E) RNA uptake assay I ( Fig. 2A ) was performed using isolated lysosomes derived from SIDT2 knockdown or control siRNA-transfected cells. Relative levels of RNA uptake were quantified. Mean ± SEM ( n = 3). ***, P

    Journal: Autophagy

    Article Title: Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes

    doi: 10.1080/15548627.2016.1145325

    Figure Lengend Snippet: Effects of SIDT2 knockdown on RNA uptake and degradation by lysosomes. (A and D) Decreased levels of SIDT2 proteins in HeLa cells transfected with SIDT2 -siRNA were confirmed by immunoblotting. Relative levels of SIDT2 were quantified. Results are expressed as mean ± SEM ( n = 3). (B and E) RNA uptake assay I ( Fig. 2A ) was performed using isolated lysosomes derived from SIDT2 knockdown or control siRNA-transfected cells. Relative levels of RNA uptake were quantified. Mean ± SEM ( n = 3). ***, P

    Article Snippet: The primary antibodies used for immunoprecipitation or immunoblotting were: polyclonal goat anti-SIDT2 (N-20; Santa Cruz Biotechnology, sc-54151), polyclonal goat anti-SIDT1 (GeneTex, GTX88799), monoclonal rat anti-LAMP2 (M3/84; Santa Cruz Biotechnology, sc-19991), monoclonal mouse anti-DYKDDDDK (FLAG; Wako Pure Chemical Industries, 018-22783), monoclonal rabbit anti-SCARB2/LIMP2 (EPR12080; Abcam, ab176317), monoclonal mouse anti-RAB7A (Rab7-117; Abcam, ab50533), monoclonal mouse anti-RAB5A (RAB5A, member RAS oncogene family) (D-11; Santa Cruz Biotechnology, sc-46692), monoclonal mouse anti-MAP1LC3A/B (4E12; MBL, M152-3), polyclonal rabbit anti-LMNA (lamin A) (H-102; Santa Cruz Biotechnology, sc-20680), monoclonal mouse anti-COX4I1 (cytochrome c oxidase subunit IV isoform 1) (20E8C12; Life Technologies, A21348), monoclonal mouse anti-GOLGA1/Golgin-97 (CDF4; Life Technologies, A-21270), polyclonal rabbit anti-CANX (calnexin) (Abcam, ab22595), monoclonal mouse anti-GAPDH (6C5; Merck Millipore, CB1001), polyclonal rabbit anti-TUBB (tubulin, β)/β-tubulin (Cell Signaling Technology, 2146), and monoclonal mouse anti-ACTB/β-actin (AC-15; Sigma-Aldrich, A5441).

    Techniques: Transfection, Isolation, Derivative Assay