Journal: Physiological Reports

Article Title: Regulation of PPARGC1A gene expression in trained and untrained human skeletal muscle

doi: 10.14814/phy2.13543

Figure Lengend Snippet: Phosphorylation of exercise‐related signaling proteins and dephosphorylation of CRTC2 in exercised m. vastus lateralis prior to and at 2 min after, the one‐legged continuous knee extension exercise

Article Snippet: The proteins were transferred to nitrocellulose membranes using a Trans‐Blot Turbo system (Bio‐Rad, USA) in Towbin buffer for 30 min at 25 V. The membranes were stained with Ponceau S to verify consistent loading of proteins, followed by washing and incubation in 5% nonfat dry milk for 1 h. The membranes were subsequently incubated at 4°C overnight with antibodies specific for phospho‐acetyl‐CoA carboxylase (ACC Ser79/222 ; 1:1000; ab68191; Abcam, UK), phospho‐mitogen‐activated protein kinase (p38 Thr180/Tyr182 ; 1:500; ab195049; Abcam), phospho‐Ca 2+ /calmodulin‐dependent protein kinase II (CaMKII Thr286 ; 1:2500; ab32678; Abcam), phospho‐CREB1 Ser133 (1:500; ab32096; Abcam), CREB‐regulated transcription coactivator 2 (CRTC2; 1:500, #13017; Cell Signaling), and oxidative phosphorylation proteins (OXPHOS): NDUFB8, SDHB, UQCRC2, MT‐CO1, and ATP5A1 (1:2500; ab110413; Abcam) followed by incubation for 1 h at room temperature with an anti‐rabbit secondary antibody (Cell Signaling).

Techniques: De-Phosphorylation Assay

Journal: PLoS ONE

Article Title: The Ectodomain of TLR3 Receptor Is Required for Its Plasma Membrane Translocation

doi: 10.1371/journal.pone.0092391

Figure Lengend Snippet: Constructs used in experiments and their amino acid composition.

Article Snippet: Expression plasmids containing sequences of TLR3 (pUNO-hTLR3), TLR9 (pUNO-hTLR9-HA), and UNC93B1 (pUNO1-hUNC93B1) were from InvivoGen, TLR3-mCer (pcDNA3-hTLR3-mCerulean) containing plasmid was prepared in our lab , TLR9-YFP (pcDNA3-hTLR9-YFP) was from Addgene, plasmid constitutively expressing Renilla luciferase–phRL-TK was from Promega, pmCherry-C1 was from Clontech Laboratories.

Techniques: Construct

Journal: PLoS ONE

Article Title: The Ectodomain of TLR3 Receptor Is Required for Its Plasma Membrane Translocation

doi: 10.1371/journal.pone.0092391

Figure Lengend Snippet: (A–D) HEK293T cells were transfected with a UNC93B1-mCherry-Myc (cyan). (A) ER was dyed with ER-Tracker Blue-White DPX. (B) Lysosomes were marked with LysoTracker Green DND-26. (C) Endosomes were stained with Transferrin AlexaFluor 633 conjugate. (D) Plasma membrane was dyed with CTB-Alexa 647. All dyes are shown in magenta. White arrows indicate colocalization. (E) HEK293T cells were transfected with a UNC93B1-mCherry-Myc (magenta) and TLR3-mCer (cyan). Plasma membrane was dyed with CTB-Alexa 647 (red). Membrane localization was evaluated from plots of normalized fluorescence intensities of UNC93B1-mCherry-Myc and TLR3-mCer and plasma membrane (PM) within 3 μm line profiles (n = 9). Three representative lines are marked on merged images. Images are selected from three independent experiments. Scale bars, 10 μm.

Article Snippet: Expression plasmids containing sequences of TLR3 (pUNO-hTLR3), TLR9 (pUNO-hTLR9-HA), and UNC93B1 (pUNO1-hUNC93B1) were from InvivoGen, TLR3-mCer (pcDNA3-hTLR3-mCerulean) containing plasmid was prepared in our lab , TLR9-YFP (pcDNA3-hTLR9-YFP) was from Addgene, plasmid constitutively expressing Renilla luciferase–phRL-TK was from Promega, pmCherry-C1 was from Clontech Laboratories.

Techniques: Transfection, Staining, Fluorescence

Journal: PLoS ONE

Article Title: The Ectodomain of TLR3 Receptor Is Required for Its Plasma Membrane Translocation

doi: 10.1371/journal.pone.0092391

Figure Lengend Snippet: HEK293T cells were transfected with TLR3-mCer alone (A) or cotransfected with UNC93B1 (B). Cells were stimulated with rhodamine labeled poly(I:C) (poly(I:C)-R). (B) TLR3 and poly(I:C)-R colocalization was evaluated from plots (right) of fluorescence intensities within 3 μm line profiles (n = 3; i, ii, iii). Three representative speckles where cross-sections were analyzed are marked with the white arrows on the merged image. (C–E) HEK293 cells were transfected with TLR3 alone or with UNC93B1 encoding plasmid. Cells were cotransfected with IFN-β (left) or NF-κB (right) promoter reporter plasmid and Renilla reporter plasmid. Cells were simultaneously treated with poly(I:C) (10 μg/ml) and inhibitors. Cells were treated with increasing amounts (0.2–5 μM) of cytochalasin D (abbr. CD) (C), Dynasore (abbr. Dy) (20–80 μM) (D) or bafilomycin A (abbr. BA) (0.2–2 μM) (E). 8 h after treatment luciferase activity (RLU) was measured in the cell lysates. The results are represented by mean values with SD from triplicate wells. The representative data from three experiments are shown.

Article Snippet: Expression plasmids containing sequences of TLR3 (pUNO-hTLR3), TLR9 (pUNO-hTLR9-HA), and UNC93B1 (pUNO1-hUNC93B1) were from InvivoGen, TLR3-mCer (pcDNA3-hTLR3-mCerulean) containing plasmid was prepared in our lab , TLR9-YFP (pcDNA3-hTLR9-YFP) was from Addgene, plasmid constitutively expressing Renilla luciferase–phRL-TK was from Promega, pmCherry-C1 was from Clontech Laboratories.

Techniques: Transfection, Labeling, Fluorescence, Plasmid Preparation, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: The Ectodomain of TLR3 Receptor Is Required for Its Plasma Membrane Translocation

doi: 10.1371/journal.pone.0092391

Figure Lengend Snippet: (A) Schematic representation of chimeric constructs where the transmembrane segments or cytoplasmic domains of human TLR3 and TLR9 receptors have been exchanged. (B, C) HEK293T cells were transfected with TLR3, TLR3-9-3 and TLR3-3-9 (B) or TLR9, TLR9-3-9 and TLR9-9-3 (C). Western blot was performed using anti-TLR3 or anti-HA antibodies. Anti–β-actin antibodies were used as a loading control. The representative data from three experiments are shown. (D-E) HEK293 cells were transiently transfected with TLR3, TLR9 or chimeric constructs TLR3-9-3, TLR3-3-9, TLR9-3-9, and TLR9-9-3 alone or with UNC93B1. Cells were transfected with IFN-β (D, F) or NF-κB (E, G) promoter reporter plasmids and Renilla reporter plasmid. After 18 h of stimulation with poly(I:C) (10 μg/ml) (D, E) or ODN10104 (10 μg/ml) (F, G), luciferase activity (RLU) was measured in the cell lysates. The results are represented by mean values with SD from triplicate wells. The representative data from three experiments are shown.

Article Snippet: Expression plasmids containing sequences of TLR3 (pUNO-hTLR3), TLR9 (pUNO-hTLR9-HA), and UNC93B1 (pUNO1-hUNC93B1) were from InvivoGen, TLR3-mCer (pcDNA3-hTLR3-mCerulean) containing plasmid was prepared in our lab , TLR9-YFP (pcDNA3-hTLR9-YFP) was from Addgene, plasmid constitutively expressing Renilla luciferase–phRL-TK was from Promega, pmCherry-C1 was from Clontech Laboratories.

Techniques: Construct, Transfection, Western Blot, Plasmid Preparation, Luciferase, Activity Assay

Journal: PLoS ONE

Article Title: The Ectodomain of TLR3 Receptor Is Required for Its Plasma Membrane Translocation

doi: 10.1371/journal.pone.0092391

Figure Lengend Snippet: Responsiveness and fold induction after UNC93B1 overexpression of constructs containing TLR3 and TLR9 domains.

Article Snippet: Expression plasmids containing sequences of TLR3 (pUNO-hTLR3), TLR9 (pUNO-hTLR9-HA), and UNC93B1 (pUNO1-hUNC93B1) were from InvivoGen, TLR3-mCer (pcDNA3-hTLR3-mCerulean) containing plasmid was prepared in our lab , TLR9-YFP (pcDNA3-hTLR9-YFP) was from Addgene, plasmid constitutively expressing Renilla luciferase–phRL-TK was from Promega, pmCherry-C1 was from Clontech Laboratories.

Techniques: Over Expression, Construct

Journal: PLoS ONE

Article Title: The Ectodomain of TLR3 Receptor Is Required for Its Plasma Membrane Translocation

doi: 10.1371/journal.pone.0092391

Figure Lengend Snippet: HEK293T cells were transiently transfected TLR3-9-3-mCer (A and B - cyan), TLR9-3-9-YFP (C and D - yellow), and with UNC93B1. Plasma membrane markers SynaptoRed and CTB-Alexa 555 are shown in magenta. (A) Localization of TLR3-9-3-mCer on plasma membrane in cells overexpressing UNC93B1. (B) Intracellular localization of TLR3-9-3-mCer in HEK293T without overexpression of UNC93B1. (C) Intracellular localization of TLR9-3-9-YFP in cells overexpressing UNC93B1. (D) Intracellular localization of TLR9-3-9-YFP in HEK293T without overexpression of UNC93B1. (A-D) Data are representative of three experiments. TLR membrane localization was evaluated from plots (bottom) of normalized fluorescence intensities of TLR and plasma membrane (PM) within 3 μm line profiles (n = 9). Three representative lines are marked on merged images. Images are selected from three independent experiments. Scale bars, 10 μm.

Article Snippet: Expression plasmids containing sequences of TLR3 (pUNO-hTLR3), TLR9 (pUNO-hTLR9-HA), and UNC93B1 (pUNO1-hUNC93B1) were from InvivoGen, TLR3-mCer (pcDNA3-hTLR3-mCerulean) containing plasmid was prepared in our lab , TLR9-YFP (pcDNA3-hTLR9-YFP) was from Addgene, plasmid constitutively expressing Renilla luciferase–phRL-TK was from Promega, pmCherry-C1 was from Clontech Laboratories.

Techniques: Transfection, Over Expression, Fluorescence

Journal: PLoS ONE

Article Title: The Ectodomain of TLR3 Receptor Is Required for Its Plasma Membrane Translocation

doi: 10.1371/journal.pone.0092391

Figure Lengend Snippet: HEK293T cells were transiently transfected TLR3-3-9-YFP (A and B - yellow) or TLR9-9-3-YFP (C and D - yellow) and with UNC93B1. Plasma membrane markers SynaptoRed and CTB-Alexa 555 are shown in magenta. (A) Localization of TLR3-3-9-YFP on plasma membrane in cells overexpressing UNC93B1. (B) Intracellular localization of TLR3-3-9-YFP in HEK293T without overexpression of UNC93B1. (C) Intracellular localization of TLR9-9-3-YFP in cells overexpressing UNC93B1. (D) Intracellular localization of TLR9-9-3-YFP in HEK293T without overexpression of UNC93B1. (A–D) Data are representative of three experiments. TLR membrane localization was evaluated from plots (bottom) of normalized fluorescence intensities of TLR and plasma membrane (PM) within 3 μm line profiles (n = 9). Three representative lines are marked on merged images. Images are selected from three independent experiments. Scale bars, 10 μm.

Article Snippet: Expression plasmids containing sequences of TLR3 (pUNO-hTLR3), TLR9 (pUNO-hTLR9-HA), and UNC93B1 (pUNO1-hUNC93B1) were from InvivoGen, TLR3-mCer (pcDNA3-hTLR3-mCerulean) containing plasmid was prepared in our lab , TLR9-YFP (pcDNA3-hTLR9-YFP) was from Addgene, plasmid constitutively expressing Renilla luciferase–phRL-TK was from Promega, pmCherry-C1 was from Clontech Laboratories.

Techniques: Transfection, Over Expression, Fluorescence

Journal: PLoS ONE

Article Title: The Ectodomain of TLR3 Receptor Is Required for Its Plasma Membrane Translocation

doi: 10.1371/journal.pone.0092391

Figure Lengend Snippet: HEK293T cells were transiently transfected with TLR3-mCer, TLR9-YFP, TLR3-9-3-mCer, TLR9-3-9-YFP, TLR3-3-9-YFP or TLR9-9-3-YFP. ER was dyed with ER-Tracker Red, lysosomes were marked with LysoTracker Red DND-99. To stain endosomes, cells were cotransfected with EEA1-Tomato. All dyes are shown in magenta. White arrows indicate colocalization. Images are selected from three independent experiments. Scale bars, 10 μm.

Article Snippet: Expression plasmids containing sequences of TLR3 (pUNO-hTLR3), TLR9 (pUNO-hTLR9-HA), and UNC93B1 (pUNO1-hUNC93B1) were from InvivoGen, TLR3-mCer (pcDNA3-hTLR3-mCerulean) containing plasmid was prepared in our lab , TLR9-YFP (pcDNA3-hTLR9-YFP) was from Addgene, plasmid constitutively expressing Renilla luciferase–phRL-TK was from Promega, pmCherry-C1 was from Clontech Laboratories.

Techniques: Transfection, Staining