mouse anti-mglur2  (ATS Bio)

Journal: bioRxiv

Article Title: Translation-independent association of mRNAs encoding protomers of the 5-HT 2A -mGlu2 receptor complex in living cells

doi: 10.1101/2024.06.17.599432

Figure Lengend Snippet: ( A-C ) HEK293 cells were transfected with non-targeting siRNA or 5-HT 2A R siRNA. Forty-eight hours after siRNA transfection, cells were transfected with pcDNA3.1-cMyc-5-HT 2A R. RNA and protein extractions were carried out 24 h following DNA transfection. 5-HT 2A R mRNA was assessed by RT-qPCR (n = 4 independent experiments) ( A ), and 5-HT 2A R immunoreactivity was assessed by Western blot (n = 3 independent experiments) ( B ). Representative immunoblots are shown ( C ). ( D-F ) HEK293 cells were transfected with non-targeting siRNA or 5-HT 2A R siRNA. Forty-eight hours after siRNA transfection, cells were co-transfected with pcDNA3.1-cMyc-5-HT 2A R and HA-mGluR2. RNA and protein extractions were carried out 24 h following DNA transfection. mGluR2 mRNA was assessed by RT-qPCR (n = 6 independent experiments) ( D ), and mGluR2 immunoreactivity was assessed by Western blot (n = 3 independent experiments) ( E ). Representative immunoblots are shown ( F ). ( G-I ) HEK293 cells were transfected with non-targeting siRNA or 5-HT 2A R siRNA. Forty-eight hours after siRNA transfection, cells were co-transfected with pcDNA3.1-cMyc-5-HT 2A R and HA-mGluR3. RNA and protein extractions were carried out 24 h following DNA transfection. mGluR3 mRNA was assessed by RT-qPCR (n = 9 independent experiments) ( G ), and mGluR3 immunoreactivity was assessed by Western blot (n = 3 independent experiments) ( H ). Representative immunoblots are shown ( I ). Unpaired two-tailed Student’s t -test (*p < 0.05).

Article Snippet: Immunoprecipitation of mGluR2 was performed overnight at 4°C using a mouse monoclonal anti-mGluR2 N-terminal antibody (Abcam, Catalog no. ab15672), followed by RNA isolation, DNase 1 treatment, cDNA preparation and analysis of target RNAs using RT-PCR and RT-qPCR assays (for primer pair sequences, see Table S2).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Translation-independent association of mRNAs encoding protomers of the 5-HT 2A -mGlu2 receptor complex in living cells

doi: 10.1101/2024.06.17.599432

Figure Lengend Snippet: ( A-C ) HEK293 cells were transfected with non-targeting siRNA or mGluR2 siRNA. Forty-eight hours after siRNA transfection, cells were transfected with pcDNA3.1-HA-mGluR2. RNA and protein extractions were carried out 24 h following DNA transfection. mGluR2 mRNA was assessed by RT-qPCR (n = 6 independent experiments) ( A ), and mGluR2 immunoreactivity was assessed by Western blot (n = 3 independent experiments) ( B ). Representative immunoblots are shown ( C ). ( D-F ) HEK293 cells were transfected with non-targeting siRNA or mGluR2 siRNA. Forty-eight hours after siRNA transfection, cells were co-transfected with pcDNA3.1-HA-mGluR2 and pcDNA3.1-c-Myc-5-HT 2A R. RNA and protein extractions were carried out 24 h following DNA transfection. 5-HT 2A R mRNA was assessed by RT-qPCR (n = 6 independent experiments) ( D ), and 5-HT 2A R immunoreactivity was assessed by Western blot (n = 3 independent experiments) ( E ). Representative immunoblots are shown ( F ). Unpaired two-tailed Student’s t -test (p > 0.05).

Article Snippet: Immunoprecipitation of mGluR2 was performed overnight at 4°C using a mouse monoclonal anti-mGluR2 N-terminal antibody (Abcam, Catalog no. ab15672), followed by RNA isolation, DNase 1 treatment, cDNA preparation and analysis of target RNAs using RT-PCR and RT-qPCR assays (for primer pair sequences, see Table S2).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Translation-independent association of mRNAs encoding protomers of the 5-HT 2A -mGlu2 receptor complex in living cells

doi: 10.1101/2024.06.17.599432

Figure Lengend Snippet: (A, B) Validation of cytosolic fractionation of RNP complexes using RIP Assay kit by using a cytosolic marker α-Tubulin (A) and a nuclear marker Lamin A/C (B) . (C) Immunoblot with an anti-cMyc antibody in HEK293 cells transiently transfected with pcDNA3.1-c-Myc-5-HT 2A R or pcDNA3.1-5-HT 2C R-cMyc. (D) Immunoblot with an anti-HA antibody in HEK293 cells transiently transfected with pcDNA3.1-HA-mGluR2 or pcDNA3.1-HA-mGluR3.

Article Snippet: Immunoprecipitation of mGluR2 was performed overnight at 4°C using a mouse monoclonal anti-mGluR2 N-terminal antibody (Abcam, Catalog no. ab15672), followed by RNA isolation, DNase 1 treatment, cDNA preparation and analysis of target RNAs using RT-PCR and RT-qPCR assays (for primer pair sequences, see Table S2).

Techniques: Fractionation, Marker, Western Blot, Transfection

Journal: bioRxiv

Article Title: Translation-independent association of mRNAs encoding protomers of the 5-HT 2A -mGlu2 receptor complex in living cells

doi: 10.1101/2024.06.17.599432

Figure Lengend Snippet: ( A ) Schematic representation illustrating the co-translational association of 5-HT 2A R and mGluR2 polypeptides, depicted in red and yellow, respectively, as they emerge from ribosomes shown in blue and green. The polypeptides originate from neighboring 5-HT 2A R and mGluR2 transcripts, depicted in black. The mGluR2 targeting antibody (anti-HA, magenta) used for immunoprecipitation of the mRNA/protein complex is indicated, bound to the N-terminal of mGluR2. ( B ) Schematic representation illustrating the co-translational association of 5-HT 2A R and mGluR2 polypeptides, depicted in red and yellow, respectively, as they emerge from ribosomes shown in blue and green. The polypeptides originate from neighboring 5-HT 2A R and mGluR2 transcripts, depicted in black. The 5-HT 2A R targeting antibody (anti-cMyc, dark green) used for immunoprecipitation of the mRNA/protein complex is indicated, bound to the N-terminal of 5-HT 2A R. ( C ) HEK293 cells were co-transfected with pcDNA3.1-HA-mGluR2, and pcDNA3.1-cMyc-5-HT 2A R or pcDNA3.1-5-HT 2C R-cMyc constructs, or mock. Images show representative RT-PCR products for mGluR2 , 5-HT 2A R and 5-HT 2C R transcripts from HEK293 cells before (Input) and after immuno-precipitation (IP) using an anti-HA antibody. For control, cells separately expressing the c-Myc- or HA-tagged forms were mixed. Data are representative from three independent experiments. ( D ) HEK293 cells were co-transfected with pcDNA3.1-cMyc-5-HT 2A R, and pcDNA3.1-HA-mGluR2, pcDNA3.1-HA-mGluR3 or pcDNA3.1-TAA-HA-mGluR2 constructs. Images show representative RT-PCR products for 5-HT 2A R , mGluR2 and mGluR3 transcripts from HEK293 cells before (Input) and after immuno-precipitation (IP) using an anti-cMyc antibody. For control, cells separately expressing the c-Myc- or HA-tagged forms were mixed. Data are representative from three independent experiments. ( E ) Schematic showing anti-cMyc antibody used to immunoprecipitate 5-HT 2A R protein and association of 5-HT 2A R and mGluR2 transcripts in the absence of mGluR2 protein. ( F ) Immunoblot demonstrating loss of HA-mGluR2 protein when the translation initiation site is mutated (TAA). (G, H) HEK293 cells were co-transfected with pcDNA3.1-cMyc-5-HT 2A R and pcDNA3.1-HA-mGluR2 constructs. Images show representative RT-PCR products for 5-HT 2A R and mGluR2 transcripts from HEK293 cells before (Input) and after immuno-precipitation (IP) using an anti-HA antibody. RIP assays were performed in presence and absence of EDTA (25 mM). ( H ) Quantification of change in IP band intensities of RT-PCR products for 5-HT 2A R and mGluR2 transcripts as observed in presence and absence of 25mM EDTA. Representative image ( G ), and quantification of IP/input band intensities (n = 3 independent experiments performed in triplicate) ( H ). ( I ) Images show representative RT-PCR products for 5-HT 2A R , 5-HT 2C R and mGluR2 transcripts from mouse frontal cortex samples before (Input) and after immuno-precipitation (IP) using an anti-mGluR2 antibody. Data are representative from three independent experiments in 3 mice. ( J ) Mouse frontal cortex samples were subjected to RIP assays employing an anti-mGluR2 antibody. Subsequently, the RNP complexes underwent processing for RNA isolation and RT-qPCR assays for the detection of 5-HT 2A R and 5-HT 2C R transcripts. Data are shown as fold change of IP/Input (n = 3 mice). Two-way ANOVA followed by Bonferroni’s post-hoc test ( H ), and unpaired two-tailed Student’s t -test ( J ). (*p < 0.05, **p < 0.001).

Article Snippet: Immunoprecipitation of mGluR2 was performed overnight at 4°C using a mouse monoclonal anti-mGluR2 N-terminal antibody (Abcam, Catalog no. ab15672), followed by RNA isolation, DNase 1 treatment, cDNA preparation and analysis of target RNAs using RT-PCR and RT-qPCR assays (for primer pair sequences, see Table S2).

Techniques: Immunoprecipitation, Transfection, Construct, Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Western Blot, Isolation, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: Translation-independent association of mRNAs encoding protomers of the 5-HT 2A -mGlu2 receptor complex in living cells

doi: 10.1101/2024.06.17.599432

Figure Lengend Snippet: ( A ) Confocal micrographs of HEK293 cells transiently transfected with pcDNA3.1-cMyc-5-HT 2A R-mCherry (red) and HA-mGluR2-mCitrine (green), and stained with anti-calnexin (upper panel, magenta) or anti-58-K (lower panel, magenta) and secondary antibodies, and imaged to detect mCherry, mCitrine, anti-calnexin, and anti-58-K. Nuclei were stained in blue with Hoechst. Scale bars, 5 µM. ( B ) Schematic representation of a working model for the mechanism of 5-HT 2A R and mGluR2 transcript association and subcellular localization of the 5-HT 2A R-mGluR2 heterocomplex during maturation. Following co-translational association within RNP complexes containing RPS24, 5-HT 2A R and mGluR2 are trafficked to the ER as part of a protein complex. Subsequently, this GPCR heterocomplex is translocated to the Golgi apparatus and directed to the cell membrane or other subcellular compartments.

Article Snippet: Immunoprecipitation of mGluR2 was performed overnight at 4°C using a mouse monoclonal anti-mGluR2 N-terminal antibody (Abcam, Catalog no. ab15672), followed by RNA isolation, DNase 1 treatment, cDNA preparation and analysis of target RNAs using RT-PCR and RT-qPCR assays (for primer pair sequences, see Table S2).

Techniques: Transfection, Staining, Membrane

Journal: bioRxiv

Article Title: Translation-independent association of mRNAs encoding protomers of the 5-HT 2A -mGlu2 receptor complex in living cells

doi: 10.1101/2024.06.17.599432

Figure Lengend Snippet: (A-C) Mouse frontal cortex samples were subjected to RIP assays employing an anti-mGluR2 antibody. Subsequently, input and IP samples underwent processing for RNA isolation and RT-qPCR assays for the detection of 5-HT 2A R (A) , 5-HT 2C R (B) and mGluR2 (C) transcripts (n = 3 mice). Unpaired two-tailed Student’s t -test (*p < 0.05, **p < 0.01).

Article Snippet: Immunoprecipitation of mGluR2 was performed overnight at 4°C using a mouse monoclonal anti-mGluR2 N-terminal antibody (Abcam, Catalog no. ab15672), followed by RNA isolation, DNase 1 treatment, cDNA preparation and analysis of target RNAs using RT-PCR and RT-qPCR assays (for primer pair sequences, see Table S2).

Techniques: Isolation, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: Translation-independent association of mRNAs encoding protomers of the 5-HT 2A -mGlu2 receptor complex in living cells

doi: 10.1101/2024.06.17.599432

Figure Lengend Snippet: ( A ) Venn diagram showing the overlap of identified polypeptides by LC-MS/MS in parental HEK293 cells, cells transfected solely with pcDNA3.1-HA-mGluR2 or pcDNA3.1-HA-mGluR3, and cells co-transfected with pcDNA3.1-HA-mGluR2 and pcDNA3.1-cMyc-5HT 2A R following RIP assay using the anti-HA antibody. ( B ) Dot plot depicting polypeptides present in HEK293 cells transfected solely with pcDNA3.1-HA-mGluR2 as well as in cells co-transfected with pcDNA3.1-HA-mGluR2 and pcDNA3.1-cMyc-5HT 2A R, but not in parental HEK293 cells or in cells transfected solely with pcDNA3.1-HA-mGluR3. ( C ) LC-MS/MS analysis identifying with high confidence two tryptic peptides – QMVIDVLHPGK (top) and TTPKVIFVFGFR (bottom) – from RPS24. Mass-to-Charge (m/z) peptide fragments for the y ions (blue) and the b ions (red) are displayed in the spectra (D, E) HEK293 cells were co-transfected with pcDNA3.1-cMyc-5-HT 2A R, and pcDNA3.1-HA-mGluR2 or pcDNA3.1-HA-mGluR3. Images show representative RT-PCR products for 5-HT 2A R , mGluR2 and mGluR3 transcripts from HEK293 cells before (Input) and after immuno-precipitation (IP) using an anti-RPS24 antibody. Representative image ( D ), and quantification of IP/input band intensities (n = 3 independent experiments) ( E ). ( F ) HEK293 cells were transfected with non-targeting siRNA or RPS24 siRNA. Forty-eight hours after siRNA transfection, cells were transfected with pcDNA3.1-c-Myc-5-HT 2A R and pcDNA3.1-HA-mGluR2 constructs. RIP assays were carried out 24 h following DNA transfection using an anti-HA antibody. Subsequently, the RNP complexes underwent processing for RNA isolation and RT-qPCR assays for the detection of 5-HT 2A R and mGluR2 transcripts. Data are shown as fold change of IP/Input (n = 4 independent samples). Unpaired two-tailed Student’s t -test (*p < 0.05, ***p < 0.001).

Article Snippet: Immunoprecipitation of mGluR2 was performed overnight at 4°C using a mouse monoclonal anti-mGluR2 N-terminal antibody (Abcam, Catalog no. ab15672), followed by RNA isolation, DNase 1 treatment, cDNA preparation and analysis of target RNAs using RT-PCR and RT-qPCR assays (for primer pair sequences, see Table S2).

Techniques: Liquid Chromatography with Mass Spectroscopy, Transfection, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Construct, Isolation, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: Translation-independent association of mRNAs encoding protomers of the 5-HT 2A -mGlu2 receptor complex in living cells

doi: 10.1101/2024.06.17.599432

Figure Lengend Snippet: (A) Immunoblot depicting RPS24 immunoreactivity in RIP preparations from parental HEK293 cells, as well as cells co-transfected with pcDNA3.1-cMyc-5HT 2A R, and pcDNA3.1-HA-mGluR2 or pcDNA3.1-HA-mGluR3. (B) HEK293 cells were transfected with non-targeting siRNA or RPS24 siRNA. Forty-eight hours after siRNA transfection, cells were co-transfected with pcDNA3.1-cMyc-5HT 2A R and pcDNA3.1-HA-mGluR2 constructs. RNA extractions were carried out 24 h following DNA transfection in Input samples. RPS24 mRNA was assessed by RT-qPCR (n = 4 independent samples). Unpaired two-tailed Student’s t -test (***p < 0.01).

Article Snippet: Immunoprecipitation of mGluR2 was performed overnight at 4°C using a mouse monoclonal anti-mGluR2 N-terminal antibody (Abcam, Catalog no. ab15672), followed by RNA isolation, DNase 1 treatment, cDNA preparation and analysis of target RNAs using RT-PCR and RT-qPCR assays (for primer pair sequences, see Table S2).

Techniques: Western Blot, Transfection, Construct, Quantitative RT-PCR, Two Tailed Test

Journal: Nature Microbiology

Article Title: Influenza virus uses mGluR2 as an endocytic receptor to enter cells

doi: 10.1038/s41564-024-01713-x

Figure Lengend Snippet: a , Effect of seven different membrane proteins that interact with KCa1.1 on influenza virus replication evaluated using an RNAi assay. b , mGluR2 knockdown reduced the internalization of H1N1 virus into A549 cells. c , mGluR2 knockdown reduced the internalization of H1N1 virus into A549 cells, as confirmed using a microscopy-based assay. Cell nucleus, blue; viral HA, green. d , H1N1 virus replication in A549 cells with different treatments. e , Interaction of mGluR2 and KCa1.1 confirmed by co-immunoprecipitation with agarose beads coupled with anti-Flag antibodies. f , Co-overexpression of KCa1.1 and mGluR2 increased H1N1 virus internalization. g , KCa1.1 did not internalize with H1N1 virus into A549 cells. Cell nucleus, blue; KCa1.1, green. h , mGluR2 internalized with H1N1 virus into A549 cells. Cell nucleus, blue; mGluR2, green. i , KCa1.1 knockdown prevented internalization of mGluR2 with influenza virus into A549 cells. Cell nucleus, blue; mGluR2, green. The microscopy-based assays shown in c , g , h and i were performed under unpermeabilized conditions. The solid line represents median and dashed lines represent quartiles of the data in the violin graphs of c and g – i . The images in c , e , g , h and i are representative of three independent experiments. Error bar in panels b , d and f indicates the standard deviation. The data shown in a – d and f – i are means ± s.d. ( n = 3 biologically independent experiments). Statistical analysis was performed using the unpaired, two-tailed Student’s t -test; NS, not significant. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Exact P values are available in the Source Data.

Article Snippet: Mouse anti-mGluR2 mAb (CN sc271654, 1:200) was from Santa Cruz Biotechnology.

Techniques: Membrane, Virus, RNAi Assay, Knockdown, Microscopy, Immunoprecipitation, Over Expression, Standard Deviation, Two Tailed Test

Journal: Nature Microbiology

Article Title: Influenza virus uses mGluR2 as an endocytic receptor to enter cells

doi: 10.1038/s41564-024-01713-x

Figure Lengend Snippet: a , Knockdown of CLTC significantly prevented mGluR2 internalization in H1N1-virus-internalized A549 cells. Cell nucleus, blue; mGluR2, green. b , Direct interaction of mGluR2 and HA shown using a pull-down assay with the agarose beads coupled with anti-Flag antibodies. c , Co-localization of mGluR2 and HA confirmed using a STED super-resolution microscopy assay. Cell nucleus, blue; mGluR2, red; and viral HA, green. d , mGluR2 molecules detected on the cell membrane where influenza virus attached, on the membrane of different stages of CCPs of influenza virus and on the membrane of matured CCVs of influenza virus by means of immunoelectron microscopy in mGluR2-overexpressing A549 cells. Green arrow, mGluR2; red arrow, influenza virus. Scale bar, 100 nm. e , g , mGluR2–GST ( e ) or mGluR2–ma ( g ) did not affect the viability of A549 cells. f , h , mGluR2–GST ( f ) or mGluR2–ma ( h ) treatment significantly reduced H1N1 virus replication but did not reduce adenovirus type 5 replication in A549 cells. i , Internalization of transferrin was affected by chlorpromazine (a CME inhibitor) but not by mGluR2-GST or mGluR2-ma. The microscopy-based assay shown in a and i was performed under unpermeabilized conditions and that shown in c was performed under permeabilized conditions. The solid line represents median and dashed lines represent quartiles of the data in the violin graphs of a and i . The images in a – d and i are representative of three independent experiments. Error bar in panels e – h indicates the standard deviation. The data shown in a and c – i are means ± s.d. ( n = 3 biologically independent experiments). Statistical analysis was performed using the unpaired, two-tailed Student’s t -test. NS, not significant. * P < 0.05; *** P < 0.001; **** P < 0.0001. Exact P values are available in Source Data.

Article Snippet: Mouse anti-mGluR2 mAb (CN sc271654, 1:200) was from Santa Cruz Biotechnology.

Techniques: Knockdown, Virus, Pull Down Assay, Super-Resolution Microscopy, Membrane, Immuno-Electron Microscopy, Microscopy, Standard Deviation, Two Tailed Test

Journal: Nature Microbiology

Article Title: Influenza virus uses mGluR2 as an endocytic receptor to enter cells

doi: 10.1038/s41564-024-01713-x

Figure Lengend Snippet: a , Adenovirus type 5 replication was significantly reduced in CLTC-knockdown A549 cells but not in mGluR2-knockdown A549 cells. Error bar in panels a indicates the standard deviation. The data shown in panels a is means ± s.d. (n = 3 biologically independent experiments). Statistical analysis was performed by using the unpaired, two-tailed Student’s t-test, ns, not significant, *** P < 0.001. Exact P values are available in Source Data. b , Formation of adenovirus type 5 CCPs was reduced in KCa1.1-knockdown or chlorpromazine-treated A549 cells but not in mGluR2-knockdown A549 cells. Green arrow, adenovirus type 5 CCPs; red arrow, attached adenovirus type 5. Scale bar, 200 nm. The images in b are representative of three independent experiments.

Article Snippet: Mouse anti-mGluR2 mAb (CN sc271654, 1:200) was from Santa Cruz Biotechnology.

Techniques: Knockdown, Standard Deviation, Two Tailed Test

Journal: Nature Microbiology

Article Title: Influenza virus uses mGluR2 as an endocytic receptor to enter cells

doi: 10.1038/s41564-024-01713-x

Figure Lengend Snippet: a , KCa1.1-knockdown, cyto D or paxilline treatment significantly reduced F-actin polymerization in H1N1-virus-internalized A549 cells. Cell nucleus, blue; phalloidin, green. b , Cyto D treatment or KCa1.1 knockdown prevented the influenza-virus-associated internalization of mGluR2 into A549 cells. Cell nucleus, blue; mGluR2, red. c , Cyto D or KCa1.1-knockdown treatment significantly reduced the internalization of H1N1 virus into A549 cells. d , KCa1.1 or mGluR2 knockdown and chlorpromazine treatment reduced the formation of CCPs of influenza virus in A549 cells. Green arrow, influenza virus CCP; red arrows, influenza virus attached. Scale bar, 200 nm. e , Model of the roles of mGluR2 and KCa1.1 in the CME of influenza virus. The microscopy-based assays shown in a and b were performed under unpermeabilized conditions. The solid line represents median and dashed lines represent quartiles of the data in the violin graphs of a and b . The images in a , b and d are representative of three independent experiments. Error bar in panels c indicates the standard deviation. The data shown in a – c are means ± s.d. ( n = 3 biologically independent experiments). Statistical analysis was performed using the unpaired, two-tailed Student’s t -test. NS, not significant. * P < 0.05; **** P < 0.0001. Exact P values are available in Source Data.

Article Snippet: Mouse anti-mGluR2 mAb (CN sc271654, 1:200) was from Santa Cruz Biotechnology.

Techniques: Knockdown, Virus, Microscopy, Standard Deviation, Two Tailed Test

Journal: Nature Microbiology

Article Title: Influenza virus uses mGluR2 as an endocytic receptor to enter cells

doi: 10.1038/s41564-024-01713-x

Figure Lengend Snippet: a and b , Replication of H5N6 ( a ) and H7N9 ( b ) was significantly impaired in mGluR2-silenced A549 cells. c and d , Internalization of H5N6 ( c ) and H7N9 ( d ) viruses in mGluR2-knockdown A549 cells was significantly lower than that for control cells. e , Interaction of H5 HA and mGluR2 demonstrated by co-immunoprecipitation with the anti-Flag antibody coupled agarose beads. f , Interaction of H7 HA and mGluR2 demonstrated by co-immunoprecipitation with the anti-Flag antibody coupled agarose beads. g , Soluble protein mGluR2-GST treatment significantly reduced H5N6 virus replication in A549 cells. h , Soluble protein mGluR2-GST treatment significantly reduced H7N9 virus replication in A549 cells. i , mGluR2-ma treatment significantly reduced H5N6 virus replication in A549 cells. j , mGluR2-ma treatment significantly reduced H7N9 virus replication in A549 cells. The images in i and j are representative of three independent experiments. Error bar in panels a – d and g – j indicates the standard deviation. The data shown in panels a – d and g – j are means ± s.d. (n = 3 biologically independent experiments). Statistical analysis was performed by using the unpaired, two-tailed Student’s t-test, * P < 0.05, ** P < 0.01, *** P < 0.001. Exact P values are available in Source Data.

Article Snippet: Mouse anti-mGluR2 mAb (CN sc271654, 1:200) was from Santa Cruz Biotechnology.

Techniques: Knockdown, Control, Immunoprecipitation, Virus, Standard Deviation, Two Tailed Test

Journal: Nature Microbiology

Article Title: Influenza virus uses mGluR2 as an endocytic receptor to enter cells

doi: 10.1038/s41564-024-01713-x

Figure Lengend Snippet: a – i , Groups of 16 wild-type C57BL/6J mice and mGluR2 − / − mice were intranasally inoculated with 10 MLD 50 of H1N1 virus ( a , b , c ), H5N6 virus ( d , e , f ) or H7N9 virus ( g , h , i ). a , d , g , Three mice in each group were euthanized on days 3 and 5 p.i., respectively, and their organs indicated were collected for virus titration in cells. b , c,e , f , h , i , The remaining 10 mice in each group were evaluated for body weight change ( b , e , h ) and survival ( c , f , i ) for 2 weeks. Error bar in panels a , b , d , e , g and h indicates the standard deviation. Data in a , d and g are means ± s.d. Statistical analysis was performed using the unpaired, two-tailed Student’s t -test. The log-rank (Mantel–Cox) test was used to analyse the statistical difference in survival rates between the wild-type and mGluR2 −/ − mice. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. PFU, plaque-forming unit; EID 50 , 50% egg infectious dose. Exact P values are available in Source Data.

Article Snippet: Mouse anti-mGluR2 mAb (CN sc271654, 1:200) was from Santa Cruz Biotechnology.

Techniques: Virus, Titration, Standard Deviation, Two Tailed Test

Journal: Nature Microbiology

Article Title: Influenza virus uses mGluR2 as an endocytic receptor to enter cells

doi: 10.1038/s41564-024-01713-x

Figure Lengend Snippet: Viral antigen in the lungs of wild-type and mGluR2 -/- mice that were euthanized on day 3 p.i. intranasally infected with 10 MLD 50 of H1N1 virus ( a ), H5N6 virus ( b ), or H7N9 virus ( c ) were detected by means of immunohistochemical staining. Scale bar, 500 μm. The images in a – c are representative of three independent experiments.

Article Snippet: Mouse anti-mGluR2 mAb (CN sc271654, 1:200) was from Santa Cruz Biotechnology.

Techniques: Infection, Virus, Immunohistochemical staining, Staining

Journal: Nature Microbiology

Article Title: Influenza virus uses mGluR2 as an endocytic receptor to enter cells

doi: 10.1038/s41564-024-01713-x

Figure Lengend Snippet: a , Schematic representation of glycosylation mutation sites in the ectodomain of mGluR2. b and c , Interaction between viral HA and mGluR2 or mGluR2-mutant was analyzed by using co-immunoprecipitation ( b ) and pull-down ( c ) with the anti-Flag antibody coupled agarose beads. d , Overexpression of pmGluR2-mutant restored influenza virus infection of mGluR2-knockdown A549 cells but not fully. e and f , Abundance of mGluR2 and mGluR2-mutant in whole cells and on the plasma membrane confirmed by western blotting ( e ) and flow cytometry ( f ). Control loadings in panel e are run on different gels, the samples derive from the same experiment and that blots were processed in parallel. The images in b , c , and e are representative of three independent experiments. Error bar in panels d and f indicates the standard deviation. The data shown in panels d – f are means ± s.d. (n = 3 biologically independent experiments). Statistical analysis was performed by using the unpaired, two-tailed Student’s t-test, ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Exact P values are available in Source Data.

Article Snippet: Mouse anti-mGluR2 mAb (CN sc271654, 1:200) was from Santa Cruz Biotechnology.

Techniques: Glycoproteomics, Mutagenesis, Immunoprecipitation, Over Expression, Virus, Infection, Knockdown, Clinical Proteomics, Membrane, Western Blot, Flow Cytometry, Control, Standard Deviation, Two Tailed Test

Journal: PLoS Pathogens

Article Title: Metabotropic glutamate receptor subtype 2 is a cellular receptor for rabies virus

doi: 10.1371/journal.ppat.1007189

Figure Lengend Snippet: (A) Schematic for mGluR2 protein. Transfection with siRNA s6195 resulted in the downregulation of mGluR2 mRNA in transfected HEK293 cells (B), SK cells (C), and N2a cells (D). Silencing of mGluR2 inhibited ERA-eGFP infection of HEK293 cells (E), SK cells (F), and N2a cells (G). NT, non-targeting siRNA. (H) Overexpression of mGluR2 enhanced the growth of ERA-eGFP at a multiplicity of infection (MOI) of five in transfected HEK293 cells; virus titers in the cell culture supernatant were determined as focus forming units (FFU) in BSR-T7/5 cells. A one-way ANOVA was used for the statistical analysis. *, p <0.05. **, p <0.01.

Article Snippet: The primary antibodies used in this study were mouse anti-mGluR2 antibody (sc271654; Santa Cruz Biotechnology, CA, USA), rabbit anti-mCherry antibody (ab183628, Abcam, Cambridge, UK), rabbit anti-Rab5 antibody (3547, Cell Signaling Technology, MA, USA), rabbit anti-Rab7 antibody (ab137029, Abcam, Cambridge, UK), rabbit anti-Histone H3 antibody (4499, Cell Signaling Technology, MA, USA), rabbit anti-Tomm20 antibody (ab186734, Abcam, Cambridge, UK), rabbit anti-alpha tubulin antibody (ab179484, Abcam, Cambridge, UK), and rabbit anti-AIF antibody (ab32516, Abcam, Cambridge, UK).

Techniques: Transfection, Infection, Over Expression, Virus, Cell Culture

Journal: PLoS Pathogens

Article Title: Metabotropic glutamate receptor subtype 2 is a cellular receptor for rabies virus

doi: 10.1371/journal.ppat.1007189

Figure Lengend Snippet: The mGluR2-Flag interacted with ERAG-Myc (A), CVSG-Myc (B), GX/09G-Myc (C), and WCBVG-Myc but not VSVG-Myc (D) in co-IP assays with plasmid-transfected HEK293 cell lysates. Purified mGluR2-GST pulled down purified ERAG-His (E). Purified mGluR2-GST pulled down ERAG-Myc, CVSG-Myc, and GX/09G-Myc but not VSVG-Myc from lysates of transfected HEK293 cells (F).

Article Snippet: The primary antibodies used in this study were mouse anti-mGluR2 antibody (sc271654; Santa Cruz Biotechnology, CA, USA), rabbit anti-mCherry antibody (ab183628, Abcam, Cambridge, UK), rabbit anti-Rab5 antibody (3547, Cell Signaling Technology, MA, USA), rabbit anti-Rab7 antibody (ab137029, Abcam, Cambridge, UK), rabbit anti-Histone H3 antibody (4499, Cell Signaling Technology, MA, USA), rabbit anti-Tomm20 antibody (ab186734, Abcam, Cambridge, UK), rabbit anti-alpha tubulin antibody (ab179484, Abcam, Cambridge, UK), and rabbit anti-AIF antibody (ab32516, Abcam, Cambridge, UK).

Techniques: Co-Immunoprecipitation Assay, Plasmid Preparation, Transfection, Purification

Journal: PLoS Pathogens

Article Title: Metabotropic glutamate receptor subtype 2 is a cellular receptor for rabies virus

doi: 10.1371/journal.ppat.1007189

Figure Lengend Snippet: The monoclonal antibody (mAb) or polyclonal antibody (pAb) against mGluR2 blocked ERA-eGFP infection of HEK293 cells (A), SK cells (B), N2a cells (C), and mPN cells (D). The mAb against mGluR2 also blocked VSV∆G-ERAG-eGFP infection (E) but failed to block VSV-eGFP infection (F) of HEK293 cells. The mAb or pAb against mGluR2 decreased the replication of ERA-eGFP in HEK293 cells (G) and mPN cells (H); virus titers in the cell culture supernatant were determined as FFU in BSR/T7-5 cells. The isotypes IgG2a and IgG at the highest concentration were used as controls for the mAb and pAb, respectively. A one-way ANOVA was used for the statistical analysis. *, p <0.05. **, p <0.01. ***, p <0.001.

Article Snippet: The primary antibodies used in this study were mouse anti-mGluR2 antibody (sc271654; Santa Cruz Biotechnology, CA, USA), rabbit anti-mCherry antibody (ab183628, Abcam, Cambridge, UK), rabbit anti-Rab5 antibody (3547, Cell Signaling Technology, MA, USA), rabbit anti-Rab7 antibody (ab137029, Abcam, Cambridge, UK), rabbit anti-Histone H3 antibody (4499, Cell Signaling Technology, MA, USA), rabbit anti-Tomm20 antibody (ab186734, Abcam, Cambridge, UK), rabbit anti-alpha tubulin antibody (ab179484, Abcam, Cambridge, UK), and rabbit anti-AIF antibody (ab32516, Abcam, Cambridge, UK).

Techniques: Infection, Blocking Assay, Virus, Cell Culture, Concentration Assay

Journal: PLoS Pathogens

Article Title: Metabotropic glutamate receptor subtype 2 is a cellular receptor for rabies virus

doi: 10.1371/journal.ppat.1007189

Figure Lengend Snippet: mGluR2-GST neutralized ERA-eGFP infection of HEK293 cells (A), SK cells (B), N2a cells (C), and mPN cells (D), and neutralized VSV∆G-ERAG-eGFP infection of HEK293 cells (E) but failed to neutralize VSV-eGFP infection of HEK293 cells (F). A one-way ANOVA was used for the statistical analysis. *, p <0.05. **, p <0.01. ***, p <0.001.

Article Snippet: The primary antibodies used in this study were mouse anti-mGluR2 antibody (sc271654; Santa Cruz Biotechnology, CA, USA), rabbit anti-mCherry antibody (ab183628, Abcam, Cambridge, UK), rabbit anti-Rab5 antibody (3547, Cell Signaling Technology, MA, USA), rabbit anti-Rab7 antibody (ab137029, Abcam, Cambridge, UK), rabbit anti-Histone H3 antibody (4499, Cell Signaling Technology, MA, USA), rabbit anti-Tomm20 antibody (ab186734, Abcam, Cambridge, UK), rabbit anti-alpha tubulin antibody (ab179484, Abcam, Cambridge, UK), and rabbit anti-AIF antibody (ab32516, Abcam, Cambridge, UK).

Techniques: Infection

Journal: PLoS Pathogens

Article Title: Metabotropic glutamate receptor subtype 2 is a cellular receptor for rabies virus

doi: 10.1371/journal.ppat.1007189

Figure Lengend Snippet: mGluR2-GST neutralized the infectivity of GX/09 street virus and protected mice from lethal virus challenges via intramuscular (A) or intracerebral (B) inoculation. The Log-rank (Mantel-Cox) test was used to analyze the statistical difference between the survival rates of the challenged mice. *, p <0.05. **, p <0.01.

Article Snippet: The primary antibodies used in this study were mouse anti-mGluR2 antibody (sc271654; Santa Cruz Biotechnology, CA, USA), rabbit anti-mCherry antibody (ab183628, Abcam, Cambridge, UK), rabbit anti-Rab5 antibody (3547, Cell Signaling Technology, MA, USA), rabbit anti-Rab7 antibody (ab137029, Abcam, Cambridge, UK), rabbit anti-Histone H3 antibody (4499, Cell Signaling Technology, MA, USA), rabbit anti-Tomm20 antibody (ab186734, Abcam, Cambridge, UK), rabbit anti-alpha tubulin antibody (ab179484, Abcam, Cambridge, UK), and rabbit anti-AIF antibody (ab32516, Abcam, Cambridge, UK).

Techniques: Infection, Virus

Journal: PLoS Pathogens

Article Title: Metabotropic glutamate receptor subtype 2 is a cellular receptor for rabies virus

doi: 10.1371/journal.ppat.1007189

Figure Lengend Snippet: Flow cytometry detected cell surface mGluR2 on RABV ERA-infected HEK293 cells (ERA), which was compared to uninfected HEK293 cells by staining with anti-mGluR2 monoclonal antibody (mGluR2) or isotype antibody control (IgG2a) at 4°C or 37°C (A). The flow cytometry results were analyzed with a one-way ANOVA (B). *, p <0.05. **, p <0.01.

Article Snippet: The primary antibodies used in this study were mouse anti-mGluR2 antibody (sc271654; Santa Cruz Biotechnology, CA, USA), rabbit anti-mCherry antibody (ab183628, Abcam, Cambridge, UK), rabbit anti-Rab5 antibody (3547, Cell Signaling Technology, MA, USA), rabbit anti-Rab7 antibody (ab137029, Abcam, Cambridge, UK), rabbit anti-Histone H3 antibody (4499, Cell Signaling Technology, MA, USA), rabbit anti-Tomm20 antibody (ab186734, Abcam, Cambridge, UK), rabbit anti-alpha tubulin antibody (ab179484, Abcam, Cambridge, UK), and rabbit anti-AIF antibody (ab32516, Abcam, Cambridge, UK).

Techniques: Flow Cytometry, Infection, Staining, Control

Journal: PLoS Pathogens

Article Title: Metabotropic glutamate receptor subtype 2 is a cellular receptor for rabies virus

doi: 10.1371/journal.ppat.1007189

Figure Lengend Snippet: N2a Cells were stained by using the Tyramide Signal Amplification immunofluorescent method. Absence of co-localization of tubulin (green), Rab7 (red), and Tomm20 (purple) served as a negative control for co-localization (A and B). Significant co-localization of the mitochondrial marker AIF (green) and Tomm20 (red) served as a positive control for co-localization (C and D). N2a cells infected with ERA-N-mCherry for 20 minutes at 37°C were used to perform immunofluorescence staining for RABV antigen (red), mGluR2 (green), Rab5 or Rab7 (purple), and the cell nuclei (blue). Co-localization of the RABV-mGluR2 complex with Rab5 (E, F) or Rab7 (H, I) was observed and counted. The images, comprising three single fluorescence channels (G, J), represent amplified random co-localization spots in the merged image within the small white box (G from E, F from J). The 3D-rendered images were generated by using Imaris software (G, J) and the co-localization of the RABV-mGluR2 complex with Rab5 (G) or Rab7 (J) from the three single fluorescence channels is indicated with the white arrowhead.

Article Snippet: The primary antibodies used in this study were mouse anti-mGluR2 antibody (sc271654; Santa Cruz Biotechnology, CA, USA), rabbit anti-mCherry antibody (ab183628, Abcam, Cambridge, UK), rabbit anti-Rab5 antibody (3547, Cell Signaling Technology, MA, USA), rabbit anti-Rab7 antibody (ab137029, Abcam, Cambridge, UK), rabbit anti-Histone H3 antibody (4499, Cell Signaling Technology, MA, USA), rabbit anti-Tomm20 antibody (ab186734, Abcam, Cambridge, UK), rabbit anti-alpha tubulin antibody (ab179484, Abcam, Cambridge, UK), and rabbit anti-AIF antibody (ab32516, Abcam, Cambridge, UK).

Techniques: Staining, Amplification, Negative Control, Marker, Positive Control, Infection, Immunofluorescence, Fluorescence, Generated, Software

Journal: PLoS Pathogens

Article Title: Metabotropic glutamate receptor subtype 2 is a cellular receptor for rabies virus

doi: 10.1371/journal.ppat.1007189

Figure Lengend Snippet: mGluR2-Flag interacted with ERAG-Myc in co-IP assays with plasmid-transfected HEK293 cell lysates at pH 5.5 and pH 7.2 (A). Purified mGluR2-GST pulled-down ERAG-Myc from the lysates of transfected HEK293 cells at pH 5.5 and pH 7.2 (B).

Article Snippet: The primary antibodies used in this study were mouse anti-mGluR2 antibody (sc271654; Santa Cruz Biotechnology, CA, USA), rabbit anti-mCherry antibody (ab183628, Abcam, Cambridge, UK), rabbit anti-Rab5 antibody (3547, Cell Signaling Technology, MA, USA), rabbit anti-Rab7 antibody (ab137029, Abcam, Cambridge, UK), rabbit anti-Histone H3 antibody (4499, Cell Signaling Technology, MA, USA), rabbit anti-Tomm20 antibody (ab186734, Abcam, Cambridge, UK), rabbit anti-alpha tubulin antibody (ab179484, Abcam, Cambridge, UK), and rabbit anti-AIF antibody (ab32516, Abcam, Cambridge, UK).

Techniques: Co-Immunoprecipitation Assay, Plasmid Preparation, Transfection, Purification