Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 and HCoV-OC43 regulate host m6A modification via activation of the mTORC1 signalling pathway to facilitate viral replication

doi: 10.1080/22221751.2024.2447620

Figure Lengend Snippet: SARS-CoV-2 enhances host m6A modification and promotes MAT2A expression. ( A ), ( B ) Caco2 cells were infected with SARS-CoV-2 at a MOI of 2. ( A ) Total RNA was extracted at 24, 48, and 72 h post-infection (hpi) and analyzed by dot blot to examine m6A modification. Methylene blue staining was used as a loading control.( B ) Expression levels of m6A machinery proteins in Caco-2 cells infected with SARS-CoV-2. ( C ) MeRIP-seq analysis identified m6A peaks in the SARS-CoV-2 genome isolated from Caco-2 cells infected with SARS-CoV-2 at a MOI of 4. ( D ) Relative abundance of SARS-CoV-2 RNA in 1 µg of total RNA from MeRIP-seq samples. ( E ) Schematic representation illustrating the relationship between MAT2A and m6A modification. ( F ) Protein levels of MAT2A and MAT2B in Caco-2 cells infected with SARS-CoV-2. The α2 and α2’ are two isoforms of the MAT2A protein. ( G ) MAT2A was knocked down in 293 T cells using short hairpin RNA (shRNA), with a non-targeted shRNA (shNC) serving as the control. ( H ), ( I ) The shNC or shMAT2A#1 293 T cell lines were cultured in DMEM containing 2% FBS for 24 h. ( H ) Levels of m6A modification in the indicated 293 T cells. ( I ) Concentrations of methionine and S-adenosylmethionine (SAM) were quantified by UPLC-MS/MS in the indicated cell lines. Data are presented as mean ± SEM of three independent experiments. Statistical significance was determined, with * P < 0.05, ** P < 0.01, and **** P < 0.0001.

Article Snippet: The primary antibodies: anti METTL3 (Proteintech, 15073-I-AP), anti METTL14 (Proteintech, 26158-1-AP), anti WTAP (Sigma-Aldrich, HPA010549), anti ALKBH5 (Sigma-Aldrich, HPA007196), anti FTO (Proteintech, 27226-1-AP), anti MAT2A (Proteintech, 55309-1-AP), MAT2B (Proteintech, 15952-1-AP), anti Phospho-p70 S6 Kinase (Thr389) (108D2) (Cell Signalling Technology, 9234 T), anti-p70 S6 Kinase (Cell Signalling Technology, 2708 T), anti Phospho-S6 Ribosomal Protein (Ser240/244) (Cell Signalling Technology, 2215S), anti S6 Ribosomal Protein (5G10) (Cell Signalling Technology, 2217S), anti 4E-BP1 (Cell Signalling Technology, 9452S), anti SARS-CoV-2-NP (Sino biological, 40588-T62), anti HCoV-OC43-NP (Sino biological, 40643-T62), anti m6A (Synaptic Systems, 202003), anti Rabbit Control IgG (Abclonal, AC005), anti IMPDH2 (Abcam, ab131158).

Techniques: Modification, Expressing, Infection, Dot Blot, Staining, Control, Isolation, shRNA, Cell Culture, Tandem Mass Spectroscopy

Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 and HCoV-OC43 regulate host m6A modification via activation of the mTORC1 signalling pathway to facilitate viral replication

doi: 10.1080/22221751.2024.2447620

Figure Lengend Snippet: SARS-CoV-2 nsp14 increases host m6A modification by inducing MAT2A expression. ( A ) Expression of MAT2A in 293 T cells transfected with varying doses of HA-nsp14 plasmid. ( B ) Immunoblots of 293 T cells transfected with plasmids encoding either wild-type nsp14, the H268A mutant, or the D331/G333A mutant. ( C ) Dot blot analysis of m6A modification in 293 T cells transfected with increasing doses of HA-nsp14 plasmid. ( D ) MeRIP-RT-qPCR analysis showing the levels of m6A in HA-nsp14 mRNA. Fold enrichment of m6A was normalized to the IgG control. ( E ), ( F ) Quantification of m6A modification in 293 T cells transfected with plasmids encoding either wild-type nsp14 or the D331/G333A mutant. ( G ) Comparative DNA sequence analysis of plasmids encoding wild-type HA-nsp14 and the D331/G333A mutant. ( H ) RT-qPCR analysis of mRNA abundance in the experiment described in panel f. The grayscale intensity was measured using Image J software. Values are presented as mean ± SEM of three independent experiments. Statistical significance was determined with * P < 0.05, ** P < 0.01, and ns indicating not significant.

Article Snippet: The primary antibodies: anti METTL3 (Proteintech, 15073-I-AP), anti METTL14 (Proteintech, 26158-1-AP), anti WTAP (Sigma-Aldrich, HPA010549), anti ALKBH5 (Sigma-Aldrich, HPA007196), anti FTO (Proteintech, 27226-1-AP), anti MAT2A (Proteintech, 55309-1-AP), MAT2B (Proteintech, 15952-1-AP), anti Phospho-p70 S6 Kinase (Thr389) (108D2) (Cell Signalling Technology, 9234 T), anti-p70 S6 Kinase (Cell Signalling Technology, 2708 T), anti Phospho-S6 Ribosomal Protein (Ser240/244) (Cell Signalling Technology, 2215S), anti S6 Ribosomal Protein (5G10) (Cell Signalling Technology, 2217S), anti 4E-BP1 (Cell Signalling Technology, 9452S), anti SARS-CoV-2-NP (Sino biological, 40588-T62), anti HCoV-OC43-NP (Sino biological, 40643-T62), anti m6A (Synaptic Systems, 202003), anti Rabbit Control IgG (Abclonal, AC005), anti IMPDH2 (Abcam, ab131158).

Techniques: Modification, Expressing, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Dot Blot, Quantitative RT-PCR, Control, Sequencing, Software

Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 and HCoV-OC43 regulate host m6A modification via activation of the mTORC1 signalling pathway to facilitate viral replication

doi: 10.1080/22221751.2024.2447620

Figure Lengend Snippet: SARS-CoV-2 nsp14 upregulates MAT2A expression by activating the mTORC1 signalling pathway. (A) Immunoblots of Caco2 cells infected with SARS-CoV-2 at a MOI of 2, analyzed at 24, 48, and 72 hpi. (B) Immunoblots of 293 T transfected with HA-nsp14 plasmid in a dose-dependent manner. (C) Transcript levels of MAT2A in 293 T cells transfected with indicated plasmids. (D) Immunoblots of 293 T cells grown overnight in the absence of serum, then treated with either vehicle (H2O) or insulin (500 nM, 30 min), or 293 T cells transfected with HA-nsp14 and treated with either vehicle (DMSO) or rapamycin (20 nM). (E – F) RAPTOR was knocked down in 293 T cells using a shRNA. (E) Immunoblots of the indicated 293 T cells transfected with HA-nsp14 in a dosedependent manner. (F) Quantification of m6A modification in the indicated 293 T cells by dot blot analysis. Values are presented as mean ± SEM of three independent experiments. Statistical significance was determined with *P < 0.05, **P < 0.01, and ns indicating not significant.

Article Snippet: The primary antibodies: anti METTL3 (Proteintech, 15073-I-AP), anti METTL14 (Proteintech, 26158-1-AP), anti WTAP (Sigma-Aldrich, HPA010549), anti ALKBH5 (Sigma-Aldrich, HPA007196), anti FTO (Proteintech, 27226-1-AP), anti MAT2A (Proteintech, 55309-1-AP), MAT2B (Proteintech, 15952-1-AP), anti Phospho-p70 S6 Kinase (Thr389) (108D2) (Cell Signalling Technology, 9234 T), anti-p70 S6 Kinase (Cell Signalling Technology, 2708 T), anti Phospho-S6 Ribosomal Protein (Ser240/244) (Cell Signalling Technology, 2215S), anti S6 Ribosomal Protein (5G10) (Cell Signalling Technology, 2217S), anti 4E-BP1 (Cell Signalling Technology, 9452S), anti SARS-CoV-2-NP (Sino biological, 40588-T62), anti HCoV-OC43-NP (Sino biological, 40643-T62), anti m6A (Synaptic Systems, 202003), anti Rabbit Control IgG (Abclonal, AC005), anti IMPDH2 (Abcam, ab131158).

Techniques: Expressing, Western Blot, Infection, Transfection, Plasmid Preparation, shRNA, Modification, Dot Blot

Journal: Emerging Microbes & Infections

Article Title: SARS-CoV-2 and HCoV-OC43 regulate host m6A modification via activation of the mTORC1 signalling pathway to facilitate viral replication

doi: 10.1080/22221751.2024.2447620

Figure Lengend Snippet: β-Coronaviruses stimulate SAM synthesis through the mTORC1 pathway to facilitate viral replication. ( A ) Caco2 cells were infected with HCoV-OC43, and total RNA was measured for m6A modification by dot blot analysis. ( B ) Immunoblot of Caco2 cells infected with HCoV-OC43. ( C ) Immunoblot of 293 T cells transfected with plasmids encoding SARS-CoV-1, SARS-CoV-2, and HCoV-OC43 nsp14. ZIKV-NS5 (an N7-MTase) was used as a negative control. ( D ) Immunoblot of MAT2A knockdown Caco2 cells infected with HCoV-OC43. ( E ) – ( H ) Caco2 and HRT-18 cells were cultured in the absence of methionine (Met-free). ( E ) Immunoblot of Caco2 cells infected with HCoV-OC43 with L-methionine (L-Met) supplemented in a dose-dependent manner. ( F ) Immunoblot of HRT-18 cells treated as in ( E ). ( G ) Immunoblot of Caco2 cells infected with HCoV-OC43 with SAM supplemented in a dose-dependent manner. ( H ) Immunoblot of HRT-18 cells treated as in ( G ). Values are presented as mean ± SEM of three independent experiments. Statistical significance was determined with * P < 0.05, ** P < 0.01, and ns indicating not significant.

Article Snippet: The primary antibodies: anti METTL3 (Proteintech, 15073-I-AP), anti METTL14 (Proteintech, 26158-1-AP), anti WTAP (Sigma-Aldrich, HPA010549), anti ALKBH5 (Sigma-Aldrich, HPA007196), anti FTO (Proteintech, 27226-1-AP), anti MAT2A (Proteintech, 55309-1-AP), MAT2B (Proteintech, 15952-1-AP), anti Phospho-p70 S6 Kinase (Thr389) (108D2) (Cell Signalling Technology, 9234 T), anti-p70 S6 Kinase (Cell Signalling Technology, 2708 T), anti Phospho-S6 Ribosomal Protein (Ser240/244) (Cell Signalling Technology, 2215S), anti S6 Ribosomal Protein (5G10) (Cell Signalling Technology, 2217S), anti 4E-BP1 (Cell Signalling Technology, 9452S), anti SARS-CoV-2-NP (Sino biological, 40588-T62), anti HCoV-OC43-NP (Sino biological, 40643-T62), anti m6A (Synaptic Systems, 202003), anti Rabbit Control IgG (Abclonal, AC005), anti IMPDH2 (Abcam, ab131158).

Techniques: Infection, Modification, Dot Blot, Western Blot, Transfection, Negative Control, Knockdown, Cell Culture

Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: Primer sequences used for real-time PCR.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Sequencing

Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: MAT2A expression was upregulated in inflamed gingival tissues.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Expressing

Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: MAT2A inhibition decreased P. gingivalis -induced inflammatory responses in hGfs.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Inhibition

Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: MAT2A-knockdown reduced inflammation in P. gingivalis -infected hGFs.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Knockdown, Infection

Journal: Journal of Oral Microbiology

Article Title: MAT2A inhibition suppresses inflammation in Porphyromonas gingivalis -infected human gingival fibroblasts

doi: 10.1080/20002297.2023.2292375

Figure Lengend Snippet: NF-κB/MAPK pathway was involved in the enhanced MAT2A expression.

Article Snippet: The cells were seeded on six-well plates overnight, pretreated with the MAT2A inhibitor PF9366 (S0435; Selleck Chemicals, USA), SAM (S5109; Selleck Chemicals, USA), or small interference RNA transfection if necessary, followed by incubation with antibiotic-free DMEM and 10% FBS, and at a density of 2 × 10 5 cells/2 mL per well before infection.

Techniques: Expressing

Journal: Cell Death Discovery

Article Title: Knockdown of METTL16 disrupts learning and memory by reducing the stability of MAT2A mRNA

doi: 10.1038/s41420-022-01220-0

Figure Lengend Snippet: A Overall levels of m 6 A in the hippocampi of the AAV-control ( n = 3) and AAV-sh-METTL16 group ( n = 3) were tested by m 6 A colorimetry. B Overall levels of m 6 A in the HT22 cells of the Lenti-control group ( n = 4) and Lenti-sh-METTL16-3 group ( n = 3) were tested by m 6 A colorimetry. C Expression level of MAT2A mRNA was tested by qRT–PCR in the hippocampi of MWM-trained ( n = 6) and untrained mice ( n = 6). D , E Representative images ( D ) and quantification of MAT2A ( E ) in the hippocampi of MWM-trained ( n = 5) and untrained mice ( n = 5) tested by western blotting. F , G Representative IHC images ( F ) and quantification of MAT2A ( G ) in the hippocampi of MWM-trained ( n = 3) and untrained mice ( n = 3); Scale bars = 500 μm. H Expression level of MAT2A mRNA was tested by qRT–PCR in the hippocampi of the AAV-control group ( n = 3) and AAV-sh-METTL16 group mice ( n = 3). I , J Representative images ( I ) and quantification of MAT2A ( J ) in the hippocampi of the AAV-control group ( n = 5) and AAV-sh-METTL16 group mice ( n = 5) tested by western blotting. K , L Representative IHC images ( K ) and quantification of MAT2A ( L ) in the hippocampi of the AAV-control group ( n = 5) and AAV-sh-METTL16 group mice ( n = 5); Scale bars = 500 μm. M Expression level of MAT2A mRNA was tested by qRT–PCR in the HT22 cells of the Lenti-control group ( n = 3) and Lenti-sh-METTL16-3 group ( n = 3). N , O Representative images ( N ) and quantification of MAT2A ( O ) in the HT22 cells of the Lenti-control group ( n = 4) and Lenti-sh-METTL16-3 group ( n = 4) tested by western blotting. P Representative image of stereotactic injections of AAV-sh-M16+OE-MAT2A in the hippocampi; Scale bars = 500 μm. Q , R Representative images ( Q ) and quantification of MAT2A ( R ) in the hippocampi of the AAV-sh-M16+ctrl group ( n = 5) and AAV-sh-M16+OE-MAT2A group mice ( n = 5) tested by western blotting. S , T Representative IHC images ( S ) and quantification of MAT2A ( T ) in the hippocampi of the AAV-sh-M16+ctrl group ( n = 5) and AAV-sh-M16+OE-MAT2A group mice ( n = 5); Scale bars = 500 μm. U Overexpression efficiency of MAT2A in the hippocampi was detected by qRT–PCR ( n = 4). V Overall levels of m 6 A in the hippocampi of the AAV-sh-M16+ctrl group ( n = 3) and AAV-sh-M16+OE-MAT2A group mice ( n = 3) tested by m 6 A colorimetry. W , X Representative images ( W ) and quantification of MAT2A ( X ) in the HT22 cells of the Lenti-sh-M16+vec group ( n = 4) and Lenti-sh-M16+OE-MAT2A group ( n = 4) tested by western blotting. Y Overexpression efficiency of MAT2A in the HT22 cells was detected by qRT–PCR. Z Overall levels of m 6 A in the HT22 cells of the Lenti-sh-M16+vec group ( n = 4) and Lenti-sh-M16+OE-MAT2A group ( n = 3) were tested by m 6 A colorimetry. Data shown as the mean ± SEM. P -values were determined by two-tailed t -test. * P < 0.05, ** P < 0.01.

Article Snippet: The membranes were then incubated with the following primary antibodies: rabbit anti-METTL16 (1:1,000, #17676, Cell Signaling Technology, USA), rabbit anti-METTL16 (1:1,000, #TA504710, ORIGENE, USA), rabbit anti-MAT2A (1:1,000, #NBP1-92100, Novus, USA), rabbit anti-Drebrin (1:2,000, #10260-1-AP, Proteintech, China), rabbit anti-PSD95 (1:2,000, #ab18258, Abcam, USA), rabbit anti-Syp (1:2,000, #CY5273, Abways, China), rabbit anti-GAPDH (1:10,000, #ab9485, Abcam, USA), or mouse anti-GAPDH (1:50,000, #60004–1-Ig, Proteintech, China) at 4 °C overnight.

Techniques: Control, Colorimetric Assay, Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Knockdown of METTL16 disrupts learning and memory by reducing the stability of MAT2A mRNA

doi: 10.1038/s41420-022-01220-0

Figure Lengend Snippet: A Representative Golgi-Cox impregnated spines in the hippocampi of the AAV-sh-M16+ctrl and AAV-sh-M16+OE-MAT2A group; Scale bars = 10 μm. B , C Quantification of the spine density ( B ) and the percentage of tin spine, filopodia spine, mushroom-like spine, and stubby spine ( C ) in the hippocampi of the two groups of mice ( n = 36 neurons from 6 mice per group). D Quantification of the spine density of mushroom-like and stubby spine in the hippocampi of the two groups of mice ( n = 36 neurons from 6 mice per group). E , F Representative images ( E ) and quantification of Drebrin, PSD95, and Syp ( F ) in the hippocampi of the two groups of mice were tested by western blotting ( n = 5). G , H Representative IHC images ( G ) and quantification of Drebrin, PSD95, and Syp ( H ) in the hippocampi of the two groups of mice ( n = 5); Scale bars = 500 μm. I , J Representative images ( I ) and quantification of Drebrin, PSD95, and Syp ( J ) in the HT22 cells of the Lenti-sh-M16+vec group ( n = 4) and Lenti-sh-M16+OE-MAT2A group ( n = 4) tested by western blotting. K – N Entries in new arm ( K ), time in new arm ( L ), latency 1st entrance to new arm ( M ), and total distance ( N ) of mice in the AAV-sh-M16+ctrl ( n = 9) and AAV-sh-M16+OE-MAT2A group ( n = 9) were tested in Y-maze test. O – Q Discrimination index (DI) for new objects of mice in the AAV-sh-M16+ctrl ( n = 9) and AAV-sh-M16+OE-MAT2A group ( n = 9) was tested in 2 h ( P ) and 24 h ( Q ) after excluding the mice’s preference for object location ( O ) in the ORT experiment. R , S Average velocity ( R ) and latency to find the platform ( S ) of mice in the AAV-sh-M16+ctrl ( n = 9) and AAV-sh-M16+OE-MAT2A group ( n = 9) were explored in the visible platform phase of MWM. T Latency to find the platform of mice in the AAV-sh-M16+ctrl ( n = 9) and AAV-sh-M16+OE-MAT2A group ( n = 9) were explored in the training trials phase of MWM. U , V Number of platform crossings ( U ) and time in target quadrants ( V ) of mice in the AAV-sh-M16+ctrl ( n = 9) and AAV-sh-M16+OE-MAT2A group ( n = 9) were explored in the probe trial phase of MWM. Data shown as the mean ± SEM. P- values were determined by two-tailed t -test ( B – D , F , H , J – S , U , V ), and two-way repeated-measures ANOVA ( T ). * P < 0.05, ** P < 0.01.

Article Snippet: The membranes were then incubated with the following primary antibodies: rabbit anti-METTL16 (1:1,000, #17676, Cell Signaling Technology, USA), rabbit anti-METTL16 (1:1,000, #TA504710, ORIGENE, USA), rabbit anti-MAT2A (1:1,000, #NBP1-92100, Novus, USA), rabbit anti-Drebrin (1:2,000, #10260-1-AP, Proteintech, China), rabbit anti-PSD95 (1:2,000, #ab18258, Abcam, USA), rabbit anti-Syp (1:2,000, #CY5273, Abways, China), rabbit anti-GAPDH (1:10,000, #ab9485, Abcam, USA), or mouse anti-GAPDH (1:50,000, #60004–1-Ig, Proteintech, China) at 4 °C overnight.

Techniques: Western Blot, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Knockdown of METTL16 disrupts learning and memory by reducing the stability of MAT2A mRNA

doi: 10.1038/s41420-022-01220-0

Figure Lengend Snippet: A , B HT22 cells were treated with 5 μg/ml ActD for up to 8 h with or without prior knockdown of METTL16. The expression levels of MAT2A mRNA ( A ) and U1 snRNA ( B ) were measured on the 0, 2, 4, 6, and 8 h after the ActD addition ( n = 3). C , D RIP-qRT–PCR was employed to investigate the direct interaction of METTL16 protein and MAT2A mRNA in the HT22 cells. Immunoblot analysis of METTL16 was performed as a control ( C ). MAT2A mRNA was assessed by qRT–PCR in endogenous METTL16, or IgG (negative control) immunoprecipitates from HT22 cells and are shown as percentages of input RNA ( n = 3) ( D ). E – G Anti-m 6 A RIP-qRT–PCR using total RNA from HT22 cells with or without METTL16 knockdown. Immunoblot analysis of m 6 A was performed as a control ( E ). Cartoon render of the PCR products in MAT2A mRNA ( F ). The indicated transcripts of MAT2A mRNA ( F ), β-actin, and U1 snRNA were quantified by qRT–PCR and are shown as percentages of input RNA ( G ) ( n = 3). H , I HT22 cells were treated with 5 μg/ml ActD for up to 8 h with prior overexpression of MAT2A mRNA-3′UTR-wild or MAT2A mRNA-3′UTR-mut. The expression levels of MAT2A mRNA ( H ) and U1 snRNA ( I ) were measured on the 0, 2, 4, 6, and 8 h after the ActD addition ( n = 3). Data shown as the mean ± SEM. P -values were determined by two-tailed t -test ( D , G ) and two-way repeated-measures ANOVA ( A , B , H , I ). * P < 0.05, ** P < 0.01.

Article Snippet: The membranes were then incubated with the following primary antibodies: rabbit anti-METTL16 (1:1,000, #17676, Cell Signaling Technology, USA), rabbit anti-METTL16 (1:1,000, #TA504710, ORIGENE, USA), rabbit anti-MAT2A (1:1,000, #NBP1-92100, Novus, USA), rabbit anti-Drebrin (1:2,000, #10260-1-AP, Proteintech, China), rabbit anti-PSD95 (1:2,000, #ab18258, Abcam, USA), rabbit anti-Syp (1:2,000, #CY5273, Abways, China), rabbit anti-GAPDH (1:10,000, #ab9485, Abcam, USA), or mouse anti-GAPDH (1:50,000, #60004–1-Ig, Proteintech, China) at 4 °C overnight.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Control, Negative Control, Over Expression, Two Tailed Test

Journal: Cell Death Discovery

Article Title: Knockdown of METTL16 disrupts learning and memory by reducing the stability of MAT2A mRNA

doi: 10.1038/s41420-022-01220-0

Figure Lengend Snippet: The primers used in qRT–PCR experiments.

Article Snippet: The membranes were then incubated with the following primary antibodies: rabbit anti-METTL16 (1:1,000, #17676, Cell Signaling Technology, USA), rabbit anti-METTL16 (1:1,000, #TA504710, ORIGENE, USA), rabbit anti-MAT2A (1:1,000, #NBP1-92100, Novus, USA), rabbit anti-Drebrin (1:2,000, #10260-1-AP, Proteintech, China), rabbit anti-PSD95 (1:2,000, #ab18258, Abcam, USA), rabbit anti-Syp (1:2,000, #CY5273, Abways, China), rabbit anti-GAPDH (1:10,000, #ab9485, Abcam, USA), or mouse anti-GAPDH (1:50,000, #60004–1-Ig, Proteintech, China) at 4 °C overnight.

Techniques:

Journal: Cell Death Discovery

Article Title: Knockdown of METTL16 disrupts learning and memory by reducing the stability of MAT2A mRNA

doi: 10.1038/s41420-022-01220-0

Figure Lengend Snippet: The sequence of wild and mutant-MAT2A mRNA-3′UTR.

Article Snippet: The membranes were then incubated with the following primary antibodies: rabbit anti-METTL16 (1:1,000, #17676, Cell Signaling Technology, USA), rabbit anti-METTL16 (1:1,000, #TA504710, ORIGENE, USA), rabbit anti-MAT2A (1:1,000, #NBP1-92100, Novus, USA), rabbit anti-Drebrin (1:2,000, #10260-1-AP, Proteintech, China), rabbit anti-PSD95 (1:2,000, #ab18258, Abcam, USA), rabbit anti-Syp (1:2,000, #CY5273, Abways, China), rabbit anti-GAPDH (1:10,000, #ab9485, Abcam, USA), or mouse anti-GAPDH (1:50,000, #60004–1-Ig, Proteintech, China) at 4 °C overnight.

Techniques: Sequencing, Mutagenesis