Journal: Neurochemistry international

Article Title: Red nucleus mGluR2 but not mGluR3 mediates inhibitory effect in the development of SNI-induced neuropathological pain by suppressing the expressions of TNF-α and IL-1β.

doi: 10.1016/j.neuint.2024.105840

Figure Lengend Snippet: Fig. 1. Reduced protein expression of mGluR2 but not mGluR3 in the RN of SNI rats. A: Neuropathological pain induced by SNI (***P < 0.001, compared with sham- operated rats). B–C: Western blotting indicated that mGluR2 and mGluR3 were constitutively expressed in the RN of normal rats, mGluR2 rather than mGluR3 was reduced at 2 weeks post-SNI (n = 6 rats per group; ***P < 0.001). D–E: Immunohistochemistry indicated that mGluR2 and mGluR3 were constitutively expressed in the RN of normal rats, mGluR2 rather than mGluR3 was reduced at 2 weeks post-SNI (n = 4 rats per group; **P < 0.01). Scale bars = 50 μm.

Article Snippet: Following routine processing of tissue sections, the sections were reacted with rabbit-derived anti-rat mGluR2 antibody (1:200, Proteintech, #19956-1-AP), anti-rat mGluR3 antibody (1:300, OriGene, # AP55076SU-N), anti-rat TNF-α antibody (1:300, Proteintech, #17590-1- AP) or anti-rat IL-1β antibody (1:300, Abcam, USA, #ab9722) overnight at 4 ◦C.

Techniques: Expressing, Western Blot, Immunohistochemistry

Journal: Neurochemistry international

Article Title: Red nucleus mGluR2 but not mGluR3 mediates inhibitory effect in the development of SNI-induced neuropathological pain by suppressing the expressions of TNF-α and IL-1β.

doi: 10.1016/j.neuint.2024.105840

Figure Lengend Snippet: Fig. 2. Activation of red nucleus mGluR2 but not mGluR3 inhibits the development of SNI-induced neuropathological pain. A: Intrarubral injection of mGluR2/3 agonist LY379268 (2.0 μg) at 2 weeks post-SNI significantly attenuated neuropathological pain as compared with vehicle alone. B: Intrarubral injection of mGluR2/3 antagonist EGLU (0.9 μg) at 10 min after LY379268 delivery significantly reversed the analgesic effect of LY379268, and did not show significant difference as compared with vehicle alone. C: Intrarubral injection of mGluR3 antagonist β-NAAG (15 μg) at 10 min after LY379268 delivery did not affect the analgesic effect of LY379268, and displayed significant difference as compared with vehicle alone. D–F: Footprint test showed that intrarubral injection of LY379268, LY379268 plus EGLU or LY379268 plus β-NAAG had no impact on the movement of rats. *P < 0.05, **P < 0.01 and ***P < 0.001, compared with vehicle.

Article Snippet: Following routine processing of tissue sections, the sections were reacted with rabbit-derived anti-rat mGluR2 antibody (1:200, Proteintech, #19956-1-AP), anti-rat mGluR3 antibody (1:300, OriGene, # AP55076SU-N), anti-rat TNF-α antibody (1:300, Proteintech, #17590-1- AP) or anti-rat IL-1β antibody (1:300, Abcam, USA, #ab9722) overnight at 4 ◦C.

Techniques: Activation Assay, Injection

Journal: Neurochemistry international

Article Title: Red nucleus mGluR2 but not mGluR3 mediates inhibitory effect in the development of SNI-induced neuropathological pain by suppressing the expressions of TNF-α and IL-1β.

doi: 10.1016/j.neuint.2024.105840

Figure Lengend Snippet: Fig. 3. Blockade of red nucleus mGluR2 but not mGluR3 in normal rats induces mechanical allodynia. A: Intrarubral injection of mGluR2/3 agonist LY379268 (2.0 μg) did not change the PWT of normal rats. B: Intrarubral injection of mGluR2/3 antagonist EGLU (0.9 μg) induced a significant mechanical allodynia in normal rats as compared with vehicle alone. C: Intrarubral injection of mGluR3 antagonist β-NAAG (15 μg) did not influence the PWT of normal rats. D–F: Footprint test demonstrated that intrarubral injection of LY379268, EGLU or β-NAAG did not influence the movement of rats. *P < 0.05 and ***P < 0.001, compared with vehicle.

Article Snippet: Following routine processing of tissue sections, the sections were reacted with rabbit-derived anti-rat mGluR2 antibody (1:200, Proteintech, #19956-1-AP), anti-rat mGluR3 antibody (1:300, OriGene, # AP55076SU-N), anti-rat TNF-α antibody (1:300, Proteintech, #17590-1- AP) or anti-rat IL-1β antibody (1:300, Abcam, USA, #ab9722) overnight at 4 ◦C.

Techniques: Injection

Journal: Neurochemistry international

Article Title: Red nucleus mGluR2 but not mGluR3 mediates inhibitory effect in the development of SNI-induced neuropathological pain by suppressing the expressions of TNF-α and IL-1β.

doi: 10.1016/j.neuint.2024.105840

Figure Lengend Snippet: Fig. 4. Red nucleus mGluR2 but not mGluR3 mediates inhibitory effect in the development of SNI-induced neuropathological pain by suppressing the expression of TNF-α. A–B: Western blotting indicated that red nucleus TNF-α was elevated at 2 weeks post-SNI, intrarubral injection of mGluR2/3 agonist LY379268 (2.0 μg) at 2 weeks post-SNI inhibited the overexpression of TNF-α, this effect was reversed by mGluR2/3 antagonist EGLU (0.9 μg) instead of selective mGluR3 antagonist β-NAAG (15 μg) (n = 6 rats per group). C–D: Western blotting displayed that intrarubral injection of LY379268 (2.0 μg) did not influence the protein expression of TNF-α in normal rats, while intrarubral injection of EGLU (0.9 μg) rather than β-NAAG (15 μg) significantly enhanced the expression of TNF-α (n = 6 rats per group). E–G: Immunohistochemistry showed that red nucleus TNF-α was elevated at 2 weeks post-SNI, intrarubral injection of LY379268 at 2 weeks post-SNI inhibited the overexpression of TNF-α, this effect was reversed by EGLU instead of β-NAAG. Intrarubral injection of LY379268 did not influence the protein expression of TNF-α in normal rats, while intrarubral injection of EGLU rather than β-NAAG significantly enhanced the expression of TNF-α (n = 4 rats per group). *P < 0.05, **P < 0.01 and ***P < 0.001. Scale bars = 50 μm.

Article Snippet: Following routine processing of tissue sections, the sections were reacted with rabbit-derived anti-rat mGluR2 antibody (1:200, Proteintech, #19956-1-AP), anti-rat mGluR3 antibody (1:300, OriGene, # AP55076SU-N), anti-rat TNF-α antibody (1:300, Proteintech, #17590-1- AP) or anti-rat IL-1β antibody (1:300, Abcam, USA, #ab9722) overnight at 4 ◦C.

Techniques: Expressing, Western Blot, Injection, Over Expression, Immunohistochemistry

Journal: Neurochemistry international

Article Title: Red nucleus mGluR2 but not mGluR3 mediates inhibitory effect in the development of SNI-induced neuropathological pain by suppressing the expressions of TNF-α and IL-1β.

doi: 10.1016/j.neuint.2024.105840

Figure Lengend Snippet: Fig. 5. Red nucleus mGluR2 but not mGluR3 mediates inhibitory effect in the development of SNI-induced neuropathological pain by suppressing the expression of IL-1β. A–B: Western blotting showed that red nucleus IL-1β was elevated at 2 weeks post-SNI, intrarubral injection of mGluR2/3 agonist LY379268 (2.0 μg) at 2 weeks post-SNI inhibited the overexpression of IL-1β, this effect was reversed by mGluR2/3 antagonist EGLU (0.9 μg) instead of selective mGluR3 antagonist β-NAAG (15 μg) (n = 6 rats per group). C–D: Western blotting displayed that intrarubral injection of LY379268 (2.0 μg) did not influence the protein expression of IL-1β in normal rats, while intrarubral injection of EGLU (0.9 μg) rather than β-NAAG (15 μg) significantly enhanced the expression of IL-1β (n = 6 rats per group). E–G: Immu nohistochemistry showed that red nucleus IL-1β was elevated at 2 weeks post-SNI, intrarubral injection of LY379268 at 2 weeks post-SNI inhibited the overexpression of IL-1β, this effect was reversed by EGLU instead of β-NAAG. Intrarubral injection of LY379268 did not influence the protein expression of IL-1β in normal rats, while intrarubral injection of EGLU rather than β-NAAG significantly enhanced the expression of IL-1β (n = 4 rats per group). **P < 0.01 and ***P < 0.001. Scale bars = 50 μm.

Article Snippet: Following routine processing of tissue sections, the sections were reacted with rabbit-derived anti-rat mGluR2 antibody (1:200, Proteintech, #19956-1-AP), anti-rat mGluR3 antibody (1:300, OriGene, # AP55076SU-N), anti-rat TNF-α antibody (1:300, Proteintech, #17590-1- AP) or anti-rat IL-1β antibody (1:300, Abcam, USA, #ab9722) overnight at 4 ◦C.

Techniques: Expressing, Western Blot, Injection, Over Expression

Journal: Neurochemistry international

Article Title: Red nucleus mGluR2 but not mGluR3 mediates inhibitory effect in the development of SNI-induced neuropathological pain by suppressing the expressions of TNF-α and IL-1β.

doi: 10.1016/j.neuint.2024.105840

Figure Lengend Snippet: Fig. 6. Schematics presents the underlying mechanisms of red nucleus mGluR II in pain modulation. A: mGluR II(mGluR2 and mGluR3) were constitutively expressed in the RN of normal rats. Blockade of red nucleus mGluR2 rather than mGluR3 in normal rats induced mechanical allodynia. Red nucleus mGluR2 but not mGluR3 mediated antinociceptive effect in normal rats through negatively modulating the expressions of pro-inflammatory factors TNF-α and IL-1β. B: Red nucleus mGluR2 but not mGluR3 was reduced in SNI rats, while TNF-α and IL-1β were elevated in SNI rats. Activation of mGluR2 rather than mGluR3 alleviated SNI-induced neuropathological pain through inhibiting the expressions of TNF-α and IL-1β. Glu, glutamate.

Article Snippet: Following routine processing of tissue sections, the sections were reacted with rabbit-derived anti-rat mGluR2 antibody (1:200, Proteintech, #19956-1-AP), anti-rat mGluR3 antibody (1:300, OriGene, # AP55076SU-N), anti-rat TNF-α antibody (1:300, Proteintech, #17590-1- AP) or anti-rat IL-1β antibody (1:300, Abcam, USA, #ab9722) overnight at 4 ◦C.

Techniques: Activation Assay

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: A monoclonal antibody against mGluR2 (mGluR2-ma, 200 mg ml −1 ; Santa Cruz Biotechnology, CN sc271654) was used for the antibody blocking assay.

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: A monoclonal antibody against mGluR2 (mGluR2-ma, 200 mg ml −1 ; Santa Cruz Biotechnology, CN sc271654) was used for the antibody blocking assay.

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: A monoclonal antibody against mGluR2 (mGluR2-ma, 200 mg ml −1 ; Santa Cruz Biotechnology, CN sc271654) was used for the antibody blocking assay.

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: A monoclonal antibody against mGluR2 (mGluR2-ma, 200 mg ml −1 ; Santa Cruz Biotechnology, CN sc271654) was used for the antibody blocking assay.

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: A monoclonal antibody against mGluR2 (mGluR2-ma, 200 mg ml −1 ; Santa Cruz Biotechnology, CN sc271654) was used for the antibody blocking assay.

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: A monoclonal antibody against mGluR2 (mGluR2-ma, 200 mg ml −1 ; Santa Cruz Biotechnology, CN sc271654) was used for the antibody blocking assay.

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: A monoclonal antibody against mGluR2 (mGluR2-ma, 200 mg ml −1 ; Santa Cruz Biotechnology, CN sc271654) was used for the antibody blocking assay.

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: A monoclonal antibody against mGluR2 (mGluR2-ma, 200 mg ml −1 ; Santa Cruz Biotechnology, CN sc271654) was used for the antibody blocking assay.

Techniques: Glycoproteomics, Mutagenesis, Immunoprecipitation, Over Expression, Virus, Infection, Knockdown, Clinical Proteomics, Membrane, Western Blot, Flow Cytometry, Control, 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 , 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