Journal: Scientific Reports

Article Title: A novel family of mammalian transmembrane proteins involved in cholesterol transport

doi: 10.1038/s41598-017-07077-z

Figure Lengend Snippet: Cholesterol reduction induces the relocalization of SIDT1 to the plasma membrane. ( A ) SIDT1-GFP localization observed by confocal microscopy in HEK293 cells. The yellow line indicates the line where fluorescence was measured to detect the intensity levels of SIDT1-GFP before and after methyl beta cyclodextrin (MβCD) treatment for 40 minutes. The right panel shows the fluorescence intensity along the line before and after MβCD. Data shows mean ± standard deviation from at least 40 independent cells. Length plotted in arbitrary units (a.u.). Lower right panel shows the mean ± standard deviation as normalized fluorescence (1 is the before MβCD condition). Scale bar: 10 microns. ( B ) Co-localization of SIDT1-GFP fluorescence signal with the plasma membrane marker FM-464. ( C ) Total internal reflection fluorescence microscopy (TIRFM) representative images of the localization of SIDT1-GFP before and after MβCD treatment. The lower panel shows the mean ± standard deviation of normalized fluorescence intensity obtained from at least 40 independent experiments. Data shows initial recording time (Ti) and final recording time (Tf). Notice that Ti and Tf do not change in cells not exposed to MβCD. ( D ) Biotinylation analysis of SIDT1-GFP before and after MβCD treatment. The lower panel shows the mean ± standard deviation of biotinylated protein obtained from measuring the density of the protein bands in the gel from at least 4 independent experiments. Gels were cropped to include all conditions in a single figure and because no other bands were detected in the rest of the gel.

Article Snippet: We utilized the total internal fluorescence microscopy (TIRFM) system from TIRFLabs in microarray studies .

Techniques: Confocal Microscopy, Fluorescence, Standard Deviation, Marker, Microscopy

Journal: Scientific Reports

Article Title: A novel family of mammalian transmembrane proteins involved in cholesterol transport

doi: 10.1038/s41598-017-07077-z

Figure Lengend Snippet: The extracellular CRAC domain from SIDT1 is part of a vesiculation signal required for translocation to the plasma membrane. ( A ) Representative confocal images of HEK293 cells expressing the SIDT1 double mutant from the CRAC domains (dmSIDT1). The right panel shows the line analysis illustrating the lack of translocation of the dmSIDT1 upon MβCD treatment. The lower panel shows the mean ± standard deviation of the fluorescence intensity in the line before and after MβCD treatment obtained from at least 40 independent experiments. ( B ) Total internal reflection fluorescence microscopy (TIRFM) representative images obtained with HEK293 cells expressing the dmSIDT1. The right panel shows the mean ± standard deviation from the total fluorescence of at least 30 independent measurements. ( C ) Representative confocal images of HEK293 cells expressing the SIDT1 transmembrane mutant (tmSIDT1). Notice the normal translocation to the plasma membrane after MβCD treatment. ( D ) Total internal reflection fluorescence microscopy (TIRFM) representative images obtained with the trSIDT1 mutant. ( E ) Representative confocal images of HEK293 cells expressing the exSIDT1 mutant. Notice that this mutant does not translocate to the plasma membrane after MβCD treatment. ( F ) Total internal reflection fluorescence microscopy (TIRFM) representative images obtained with the exSIDT1 mutant. Ti, the initial time of recording and Tf, final recording time. Total fluorescence normalized to Ti. In all panels scale bar: 10 microns.

Article Snippet: We utilized the total internal fluorescence microscopy (TIRFM) system from TIRFLabs in microarray studies .

Techniques: Translocation Assay, Expressing, Mutagenesis, Standard Deviation, Fluorescence, Microscopy

Journal: Scientific Reports

Article Title: A novel family of mammalian transmembrane proteins involved in cholesterol transport

doi: 10.1038/s41598-017-07077-z

Figure Lengend Snippet: Sterol microarrays suggest that the transmembrane CRAC domain from SIDT1 binds several sterols. ( A ) Cartoon illustrating the putative secondary structure of SIDT1. The nine putative transmembrane domains are shown. In red are mark the amino acids forming the two CRAC domains and in blue a putative lysosome localization signal (not explored in the present study). ( B ) TIRFM-based microarray studies illustrating the spots containing the following sterols tested: cholesterol, cholic acid, dehydrocholic acid, β-estradiol and testosterone. As positive control, some spots in the microarray were printed with an anti-GFP antibody (lower panels). Each microarray contained 2 lines of 17 spots each. The fluorescence illustrated represents the binding of the fusion protein formed by the CRAC domain attached to GFP. For CRAC domain sequences, please refer to Material and Methods. Notice that the extracellular CRAC domain did not bind any of the sterols tested while the transmembrane CRAC domain binds to cholesterol and only weakly to cholic acid. Both fusion proteins bind strongly to the anti-GFP antibody.

Article Snippet: We utilized the total internal fluorescence microscopy (TIRFM) system from TIRFLabs in microarray studies .

Techniques: Microarray, Positive Control, Fluorescence, Binding Assay

Journal: PLoS ONE

Article Title: Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88

doi: 10.1371/journal.pone.0040773

Figure Lengend Snippet: Structure of monomeric (Compound1) and dimeric (EM-163) mimetics of the BB-loop in the TIR domain of MyD88.

Article Snippet: The open reading frame encoding the TIR domain of human MyD88 (residues 157–296) was amplified from pCDNA3-MyD88-GFP (Addgene plasmid 13026, Addgene, Cambridge, MA, USA) and inserted into pDONR201 (Invitrogen, Carlsbad, CA,).

Techniques:

Journal: PLoS ONE

Article Title: Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88

doi: 10.1371/journal.pone.0040773

Figure Lengend Snippet: A microarray-based binding assay was optimized to measure compound EM-163 binding to MyD88. Nitrocellulose coated slides were spotted in quadruplicate with anti-MyD88 antibody and MyD88 TIR domain monomer proteins using the ArrayJet Marathon printer. Biotinlyated MyD88 (25 µg/ml) was pre-incubated with compound (100 µg/ml and 50 µg/ml), dimethylsulfoxide as control, or left without pre-incubation. Streptavidin- labeled with alexa fluor 647, was added for 1 h at room temperture to detect MyD88 binding interaction. Slides analyzed using the Genepix 4000B. Data represent four similar experiments.

Article Snippet: The open reading frame encoding the TIR domain of human MyD88 (residues 157–296) was amplified from pCDNA3-MyD88-GFP (Addgene plasmid 13026, Addgene, Cambridge, MA, USA) and inserted into pDONR201 (Invitrogen, Carlsbad, CA,).

Techniques: Microarray, Binding Assay, Incubation, Control, Labeling

Journal: PLoS ONE

Article Title: Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88

doi: 10.1371/journal.pone.0040773

Figure Lengend Snippet: Increased accumulation of MyD88 in CD14 + monocytes treated with SEB in the presence of EM-163 at 1 h and 4 h. Confocal images show expression of nuclei (blue) and intracellular MyD88 (red) proteins in CD14 + monocytes. Monocytes were untreated or treated with SEB, EM-163 or SEB plus EM-163 (250 µM). Cell nuclei were labeled with DAPI (blue). Scale bar = 10 µm. Data reperesent three similar experiments with different donors.

Article Snippet: The open reading frame encoding the TIR domain of human MyD88 (residues 157–296) was amplified from pCDNA3-MyD88-GFP (Addgene plasmid 13026, Addgene, Cambridge, MA, USA) and inserted into pDONR201 (Invitrogen, Carlsbad, CA,).

Techniques: Expressing, Labeling

Journal: PLoS ONE

Article Title: Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88

doi: 10.1371/journal.pone.0040773

Figure Lengend Snippet: HEK 293T cells were co-transfected with plasmids MyD88- Flag or pCMV-HA-MyD88. Seven hours after transfection, cells were incubated for 13 h with or without −163 (500 µM to 5 µM). ( A ), Cell extracts were immunoprecipitated (IP) with anti-Flag antibody, and immune-precipitated proteins were analyzed in Western blot with anti-HA. ( B ) Densitometry analysis of the results shown in ( A ). The results are representative of three independent experiments.

Article Snippet: The open reading frame encoding the TIR domain of human MyD88 (residues 157–296) was amplified from pCDNA3-MyD88-GFP (Addgene plasmid 13026, Addgene, Cambridge, MA, USA) and inserted into pDONR201 (Invitrogen, Carlsbad, CA,).

Techniques: Transfection, Incubation, Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: Therapeutic Inhibition of Pro-Inflammatory Signaling and Toxicity to Staphylococcal Enterotoxin B by a Synthetic Dimeric BB-Loop Mimetic of MyD88

doi: 10.1371/journal.pone.0040773

Figure Lengend Snippet: EM-163 inhibition was measured by monitoring LPS-induced SEAP activity via a MyD88-mediated NF-kB driven signaling pathway. HEK 293 stable transfected cell line (TLR4-MD2-NF-kB-SEAP) was activated with LPS (TLR-4 ligand) and treated with varying concentrations of EM-163 (1 mM to 50 µM). Culture supernatants were tested for SEAP activity and compared to levels in the absence of EM-163. ( A ) Inhibition of SEAP reporter activity, ( B ) inhibition of TNF-α and IL-1β production, ( C ) Inhibition of TNF-α and IL-1β production by EM-163 after SEB stimulation of HEK 293 cells transfected with HLA-DR (α/β) and FLAG-MyD88. Results represent three experiments.

Article Snippet: The open reading frame encoding the TIR domain of human MyD88 (residues 157–296) was amplified from pCDNA3-MyD88-GFP (Addgene plasmid 13026, Addgene, Cambridge, MA, USA) and inserted into pDONR201 (Invitrogen, Carlsbad, CA,).

Techniques: Inhibition, Activity Assay, Transfection