Journal: PLoS ONE

Article Title: Synergy of Combined tPA-Edaravone Therapy in Experimental Thrombotic Stroke

doi: 10.1371/journal.pone.0098807

Figure Lengend Snippet: ( A–L ) Immunostaining performed at 1 h after tCCAo, tHI or permanent carotid ligation (pCCAo) plus hypoxia showed deposits of fibrin(ogen) and platelets (detected by anti-Integrin α2β/CD41) and P-Selectin-positive blood vessels (a marker for endothelial activation) and in the ipsilateral hemisphere following tHI insults (C, G, K). Permanent carotid ligation plus hypoxia produced the same staining pattern, but with stronger signals of all three markers (D, H, L). This staining pattern was absent in the brain after tCCAo (A, E, I) or on the contralateral hemisphere following tHI (B, F, J) (n>4 for each). ( M ) Plasminogen-zymogram showed definitive induction of the brain uPA and ∼50% increase of tPA activity in the ipsilateral hemisphere (R) at 4 h post-HI in neonates. No uPA-induction was found in adult brains at 4 h following tCCAo or tHI insults. The tPA activity in the ipsilateral hemisphere (R) following tHI was reduced to ∼67% of the level in unchallenged (UN) brains (p<0.05). The right panel is the quantification of zymogram band intensity (n = 4 for each). ( N ) The plasminogen-zymogram from plasma showed the lack of tPA activity and a similar degree of uPA activity at 4 h after tCCAo or tHI insults in adult mice. Immunoblot detection of transferrin (Tf) served as the internal control. ( O ) Direct measurement of tPA activity using a fluorescent substrate kit also showed significant reduction in the ipsilateral hemisphere at 4 h following tHI insult in adult brains (n = 3-4 for each). ( P–Q ) Immunoblot analysis showed minimal change of the tPA protein level and no sign of plasminogen activator inhibitor-1 (PAI-1) up-regulation after tCCAo or tHI insult in adult brains (n = 4 for each). Scale bar: 200 μm in A–L.

Article Snippet: The following antibodies were used for immunoblot: rabbit anti-tPA (Molecular Innovations, Novi, MI), mouse anti-PAI-1 (Molecular Innovations), and mouse anti- β-actin (Sigma, St. Louis, MO).

Techniques: Immunostaining, Ligation, Marker, Activation Assay, Produced, Staining, Activity Assay, Western Blot

Journal: Frontiers in Neurology

Article Title: Morphofunctional Organization of the Connections From the Medial and Intermediate Parts of the Central Nucleus of the Amygdala Into Distinct Divisions of the Lateral Hypothalamic Area in the Rat

doi: 10.3389/fneur.2018.00688

Figure Lengend Snippet: Photomicrographs illustrating the co-distribution of PHAL axons, MCH and Hcrt cell bodies using a triple immunofluorescence procedure. (A–C) are low magnification pictures on sections at levels 27 (in fact better seen as inter-level 27/28), 28 and 30. Note the innervation of regions containing MCH and Hcrt cell bodies and corresponding to the dorsal and suprafornical regions of the LHA. However, the ZI, or the caudal perifornical regions (level 30) that are rich in MCH cell bodies but poor in Hcrt neurons are not innervated. By contrast at level 30, the PST that contains a dense group of MCH neurons is intensely innervated. (D,E) Higher magnification illustrations showing that buttons by PHAL axons could be seen close to MCH and Hcrt cell bodies. However, it seemed to be mostly en passant putative contacts.

Article Snippet: After rinsing in PBS-T, free-floating sections were incubated with primary antibodies [anti-Hcrt (Mouse monoclonal, 1:1,000, ANGIO-PROTEOMIE®); anti-MCH [Rabbit polyclonal, 1:1,000, our laboratory , RRID:AB_2616562 ]; anti-PHAL (Goat polyclonal, 1:1,000, Vector Laboratories®, RRID:AB_10000080 )] dissolved in PBS-T, 1% bovine serum albumin, 10% lactoproteins, and 0.01% sodium azide or only in PBS-T for 24 h at 4°C.

Techniques: Immunofluorescence

Journal: Frontiers in Neurology

Article Title: Morphofunctional Organization of the Connections From the Medial and Intermediate Parts of the Central Nucleus of the Amygdala Into Distinct Divisions of the Lateral Hypothalamic Area in the Rat

doi: 10.3389/fneur.2018.00688

Figure Lengend Snippet: Photomicrographs after quadruple labeling experiments with the detection using immunofluorescence of PHAL, MCH, and Hcrt, the fourth signal being the native fluorescence of the FG. Because it is difficult to separate signals from each other on the picture, we added line drawings that schematize the distribution of these signals. Sections are from experiments PHAL#1 (A,B) or PHAL#2 (C,D) respectively, and pass approximately through the level 28. Note that the PHAL distribution is distinct in these two cases, with an innervation restricted to the LHAd in experiment PHAL#1, while the LHAs is innervated in the experiment PHAL#2. Furthermore in this experiment, innervation of FG retrogradely labeled neurons from the PSTN are observed. In fact in a few occasion we could see PHAL axons forming pericellular nets around FG-containing cell bodies lateral to the fornix [framed picture in (C) , optical slice taken using the Apotome feature of the microscope]. FG labeled neurons were observed lateral to the fornix, in an area that belong to the LHAd, and in the far ventrolateral LHA. However, we cannot rule out the possibility that the labeling of these far latero-ventral neurons could not be the result of an uptake of the FG through dendrites extending into the injection site. In the two experiments, as in the experiment AMY1, only passing axons seemed to contact MCH or Hcrt cell bodies.

Article Snippet: After rinsing in PBS-T, free-floating sections were incubated with primary antibodies [anti-Hcrt (Mouse monoclonal, 1:1,000, ANGIO-PROTEOMIE®); anti-MCH [Rabbit polyclonal, 1:1,000, our laboratory , RRID:AB_2616562 ]; anti-PHAL (Goat polyclonal, 1:1,000, Vector Laboratories®, RRID:AB_10000080 )] dissolved in PBS-T, 1% bovine serum albumin, 10% lactoproteins, and 0.01% sodium azide or only in PBS-T for 24 h at 4°C.

Techniques: Labeling, Immunofluorescence, Fluorescence, Microscopy, Injection

Journal: Journal of Virology

Article Title: Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2

doi: 10.1128/jvi.01611-22

Figure Lengend Snippet: TfR1 interacts with mGluR2. (A) Schematic representation of the AP-MS approach used to identify mGluR2-protein interactions in HEK293 cells. (B and C) HEK293 cells were co-transfected with mGluR2-Flag and TfR1-Myc, then cells were solubilized by NP-40 lysis buffer (B) or RIPA lysis buffer (C), and then subjected to immunoprecipitation (IP) by using anti-Flag agarose beads. Representative Western blots of whole cell lysates and eluates after IP are shown. (D) Co-immunoprecipitation of mGluR2-Flag with endogenous TfR1 in HEK293 cells. (E and F) Purified mGluR2-GST protein (E) or TfR1-GST protein (F) was pooled with the lysate from TfR1-Myc- (E) or mGluR2-Flag- (F) transfected HEK293 cells and then pulled down by using anti-GST beads. The GST protein was used as the negative control.

Article Snippet: The primary antibodies used in this study were: TfR1 antibody (BD Pharmingen, 555534), mCherry antibody (Abcam, ab183628), mGluR2 antibody (Abcam, ab150387), and clathrin antibody (CST, 4796S).

Techniques: Transfection, Lysis, Immunoprecipitation, Western Blot, Purification, Negative Control

Journal: Journal of Virology

Article Title: Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2

doi: 10.1128/jvi.01611-22

Figure Lengend Snippet: mGluR2, RABV, and TfR1 internalize together. (A) Increasing amounts of TfR1-GST were respectively mixed with mGluR2-Flag-conjugated beads, then the lysate from RABV G-Myc-transfected HEK293 cells was added for use in the protein interaction competition assay. (B to D) N2a cells were infected with ERA-N-mCherry for 10 min at 37°C, and then multiplex immunofluorescence was performed. The arrows indicate the colocalization of TfR1 (green), ERA-N-mCherry (red), clathrin (yellow), and mGluR2 (purple). Two representative co-localizations (scale bar, 3 μm) (yellow arrows) are shown in three dimensions (B); the dashed box is magnified at the indicated location of the same image (scale bar, 0.7 μm) (C and D).

Article Snippet: The primary antibodies used in this study were: TfR1 antibody (BD Pharmingen, 555534), mCherry antibody (Abcam, ab183628), mGluR2 antibody (Abcam, ab150387), and clathrin antibody (CST, 4796S).

Techniques: Transfection, Competitive Binding Assay, Infection, Multiplex Assay, Immunofluorescence

Journal: Journal of Virology

Article Title: Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2

doi: 10.1128/jvi.01611-22

Figure Lengend Snippet: TfR1 and TTP are required for agonist-triggered mGluR2 endocytosis. (A) HEK293 cells were treated with or without LY354740 for 30 min at 37°C, and then were stained with an antibody against mGluR2 (red). Cell nuclei were stained with Hoechst 33342. Representative images are shown. The fluorescence signal of mGluR2 was quantified by using ZEN software. The relative fluorescence of mGluR2 was quantified by normalization to control cells. The circles represent individual datapoints. At least 100 cells per sample were quantified. (B and C) TfR1-silenced (B) or TTP-silenced (C) HEK293 cells were treated with LY354740 as described in (A). Representative images are shown. The data represent the sum of three independent experiments. The two-tailed unpaired Student's t test was used for the statistical analysis. *** P < 0.001.

Article Snippet: The primary antibodies used in this study were: TfR1 antibody (BD Pharmingen, 555534), mCherry antibody (Abcam, ab183628), mGluR2 antibody (Abcam, ab150387), and clathrin antibody (CST, 4796S).

Techniques: Staining, Fluorescence, Software, Two Tailed Test

Journal: Journal of Virology

Article Title: Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2

doi: 10.1128/jvi.01611-22

Figure Lengend Snippet: TfR1 is important for the internalization of SARS-CoV-2. (A) The TfR1 mRNA level in the indicated siRNA-transfected Vero-E6 cells or Caco2 cells was measured by qPCR. (B) TfR1-silenced Vero-E6 cells or Caco2 cells were infected with SARS-CoV-2 HRB25 strain. At 24 h postinfection, virus in the culture supernatant was detected by use of plaque assays. (C and D) Co-immunoprecipitation of TfR1-Flag and SARS-CoV-2 S-Myc (C), SARS-CoV-2 S1-Myc, or SARS-CoV-2 RBD-Myc (D) in plasmid-transfected HEK293 cells. (E) Co-immunoprecipitation of TfR1-Flag and SARS-CoV-2 S-Myc in plasmid-transfected Vero-E6 cells. (F) Co-immunoprecipitation of TfR1-Flag and ACE2-Myc in plasmid-transfected HEK293 cells. (G) Cells were incubated with SARS-CoV-2 for 1 h at 4°C, then washed with acid buffer/trypsin, and lysed for qPCR to detect virus bound to the cell surface. (H and I) SARS-CoV-2 binding (H) and internalization (I) assays were performed in TfR1-silenced Vero-E6 cells or Caco2 cells. (J) Increasing amounts of mGluR2-GST were respectively mixed with TfR1-Flag-conjugated beads, and then with the lysate from SARS-CoV-2 S-Myc-transfected HEK293 cells for the protein interaction competition assay. In panels (A), (B), and (G to I), error bars represent the means ± SDs of 3 independent experiments or replicates. The two-tailed unpaired Student's t test was used for the statistical analysis. ns, not significant, * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies used in this study were: TfR1 antibody (BD Pharmingen, 555534), mCherry antibody (Abcam, ab183628), mGluR2 antibody (Abcam, ab150387), and clathrin antibody (CST, 4796S).

Techniques: Transfection, Infection, Immunoprecipitation, Plasmid Preparation, Incubation, Binding Assay, Competitive Binding Assay, Two Tailed Test

Journal: Journal of Virology

Article Title: Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2

doi: 10.1128/jvi.01611-22

Figure Lengend Snippet: TfR1 is important for SARS-CoV S- and MERS-CoV S-mediated internalization. (A and B) Co-immunoprecipitation of TfR1-Flag and SARS-CoV S-Myc (A), or MERS-CoV S-Myc (B) in plasmid-transfected HEK293 cells. (C and D) Co-immunoprecipitation of TfR1-Flag and SARS-CoV S-Myc (C), or MERS-CoV S-Myc (D) in plasmid-transfected Vero-E6 cells. (E) Cells were incubated with rVSV-SARS-CoV-S, or rVSV-MERS-CoV-S for 1 h at 4°C, then washed with acid buffer/trypsin, and lysed for qPCR to detect virus bound to the cell surface. (F and G) rVSV-SARS-CoV-S, or rVSV-MERS-CoV-S binding (F) and internalization (G) assays were performed in TfR1-silenced Vero-E6 cells. (H and I) Increasing amounts of mGluR2-GST were respectively mixed with TfR1-Flag-conjugated beads, and then with the lysate from SARS-CoV S-Myc- (H) or MERS-CoV S-Myc- (I) transfected HEK293 cells for the protein interaction competition assay. In panels (E to G), error bars represent the means ± SDs of 3 independent experiments or replicates. The two-tailed unpaired Student's t test was used for the statistical analysis. ns, not significant, ** P < 0.01; *** P < 0.001.

Article Snippet: The primary antibodies used in this study were: TfR1 antibody (BD Pharmingen, 555534), mCherry antibody (Abcam, ab183628), mGluR2 antibody (Abcam, ab150387), and clathrin antibody (CST, 4796S).

Techniques: Immunoprecipitation, Plasmid Preparation, Transfection, Incubation, Binding Assay, Competitive Binding Assay, Two Tailed Test

Journal: Journal of Virology

Article Title: Transferrin Receptor Protein 1 Cooperates with mGluR2 To Mediate the Internalization of Rabies Virus and SARS-CoV-2

doi: 10.1128/jvi.01611-22

Figure Lengend Snippet: Proposed model for the role of mGluR2 and TfR1 in RABV or SARS-CoV-2 entry. The virus first binds to a specific receptor(s) at the cell surface, then the virus interacts with and activates mGluR2. The virus-mGluR2 complex migrates to a TfR1-containing pre-existing CCP, where it interacts with TfR1 and hijacks the endocytic signaling of TfR1 to enter the cell.

Article Snippet: The primary antibodies used in this study were: TfR1 antibody (BD Pharmingen, 555534), mCherry antibody (Abcam, ab183628), mGluR2 antibody (Abcam, ab150387), and clathrin antibody (CST, 4796S).

Techniques: