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pegfp c3 vector  (TaKaRa)


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    TaKaRa pegfp c3 vector
    Pegfp C3 Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/pegfp+c3+vector/pm38940560-76-20-22
    Average 86 stars, based on 1 article reviews
    pegfp c3 vector - by Bioz Stars, 2026-09
    86/100 stars

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    Related Articles

    Clone Assay:

    Article Title: PRRSV GP5 inhibits the antivirus effects of chaperone-mediated autophagy by targeting LAMP2A.
    Article Snippet: TRIzol reagent (R401-01), HiScript II 1st Strand cDNA Synthesis Kit (R211-01), Phanta Max Super-Fidelity DNA Polymerase (P505-d1), Rapid Taq Master Mix (P222-01), and ChamQ Universal SYBR qPCR Master Mix (Q711-02) were purchased from Vazyme Biotech Co., Ltd. .. Endogenous microtubule-associated protein 1 light chain 3 (LC3), which was cloned from the PAM genome (NM_001170827), was ligated into the pEGFP-C3 vector (Clontech, 6082-1). .. GP3, GP4, GP5, M, and N, which were cloned from the PRRSV Hn07-1 Month XXXX Volume 0 Issue 0 10.1128/mbio.00532-24 3 D ow nl oa de d fr om h ttp s: //j ou rn al s. as m .o rg /jo ur na l/m bi o on 0 5 Ju ly 2 02 4 by 2 40 3: 38 00 :3 28 3: a9 e: 88 39 :6 16 f: eb 58 :7 f2 . genome (KX766378), were inserted into the pCMV-Flag-N vector (Clontech, 635688).

    Article Title: Involvement of the splicing factor SART1 in the BRCA1-dependent homologous recombination repair of DNA double-strand breaks
    Article Snippet: .. Human SART1 mRNA (NM_005146) was reverse-transcribed into complementary DNA (cDNA), which was cloned into the pEGFP-C3 vector (Clontech, Mountain View, CA, USA) using the In-Fusion HD cloning kit (Takara Bio, Shiga, Japan). .. SART1 mutants were generated using the KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan) with some modifications.

    Article Title: RNAi-Mediated c-Rel Silencing Leads to Apoptosis of B Cell Tumor Cells and Suppresses Antigenic Immune Response In Vivo
    Article Snippet: .. The PCR product was cloned into the Bgl II and EcoR I sites of pEGFP-C3 vector (Clontech) to generate a new vector, pEGFP-mU6-1 , . ..

    Article Title: Microbial metabolite n-butyrate upregulates intestinal claudin-23 expression through SP1 and AMPK pathways in mouse colon and human intestinal Caco-2 cells.
    Article Snippet: Aims: Regulation of the intestinal barrier is closely related to intestinal microbial metabolism.. This study investigated the role of intestinal microflora in the regulation of the tight junction (TJ) barrier in epithelial cells, focusing on the microbial metabolite n-butyrate, a major short-chain fatty acid, using mice and human intestinal

    Article Title: Biased exonization of transposed elements in duplicated genes: A lesson from the TIF-IA gene
    Article Snippet: .. A genomic DNA from 293T cell line (Gentra) corresponding to exon 1 through exon 2 of the TIF-IA gene (from locus 15) was PCR amplified and cloned into pEGFP-C3 vector (Clontech) between XhoI and BamHI sites under the control of the human cytomegalovirus (CMV) immediate early promoter, giving a ~1.9 kb insert. ..

    Plasmid Preparation:

    Article Title: PRRSV GP5 inhibits the antivirus effects of chaperone-mediated autophagy by targeting LAMP2A.
    Article Snippet: TRIzol reagent (R401-01), HiScript II 1st Strand cDNA Synthesis Kit (R211-01), Phanta Max Super-Fidelity DNA Polymerase (P505-d1), Rapid Taq Master Mix (P222-01), and ChamQ Universal SYBR qPCR Master Mix (Q711-02) were purchased from Vazyme Biotech Co., Ltd. .. Endogenous microtubule-associated protein 1 light chain 3 (LC3), which was cloned from the PAM genome (NM_001170827), was ligated into the pEGFP-C3 vector (Clontech, 6082-1). .. GP3, GP4, GP5, M, and N, which were cloned from the PRRSV Hn07-1 Month XXXX Volume 0 Issue 0 10.1128/mbio.00532-24 3 D ow nl oa de d fr om h ttp s: //j ou rn al s. as m .o rg /jo ur na l/m bi o on 0 5 Ju ly 2 02 4 by 2 40 3: 38 00 :3 28 3: a9 e: 88 39 :6 16 f: eb 58 :7 f2 . genome (KX766378), were inserted into the pCMV-Flag-N vector (Clontech, 635688).

    Article Title: RNAi-Mediated c-Rel Silencing Leads to Apoptosis of B Cell Tumor Cells and Suppresses Antigenic Immune Response In Vivo
    Article Snippet: .. The PCR product was cloned into the Bgl II and EcoR I sites of pEGFP-C3 vector (Clontech) to generate a new vector, pEGFP-mU6-1 , . ..

    Article Title: Microbial metabolite n-butyrate upregulates intestinal claudin-23 expression through SP1 and AMPK pathways in mouse colon and human intestinal Caco-2 cells.
    Article Snippet: Aims: Regulation of the intestinal barrier is closely related to intestinal microbial metabolism.. This study investigated the role of intestinal microflora in the regulation of the tight junction (TJ) barrier in epithelial cells, focusing on the microbial metabolite n-butyrate, a major short-chain fatty acid, using mice and human intestinal

    Article Title: STING guides the STX17-SNAP29-VAMP8 complex assembly to control autophagy
    Article Snippet: .. The STING cDNA was amplified and inserted into the p3xFLAG-Myc-CMV24 vector (Sigma) and pEGFP-C3 vector (Clontech) by HindIII and SalI to generate FLAG-STING and GFP-STING plasmids. ..

    Article Title: Biased exonization of transposed elements in duplicated genes: A lesson from the TIF-IA gene
    Article Snippet: .. A genomic DNA from 293T cell line (Gentra) corresponding to exon 1 through exon 2 of the TIF-IA gene (from locus 15) was PCR amplified and cloned into pEGFP-C3 vector (Clontech) between XhoI and BamHI sites under the control of the human cytomegalovirus (CMV) immediate early promoter, giving a ~1.9 kb insert. ..

    Article Title: Overexpression of DOC-1R Inhibits Cell Cycle G1/S Transition by Repressing CDK2 Expression and Activation
    Article Snippet: For generation of pGST-DOC-1R and its truncated mutants, DOC-1R cDNA was amplified and subcloned into a pGEX-5X-1 vector (GE Healthcare, Piscataway, NJ). .. In addition, the CDK2 coding sequence was subcloned into a pDsRED-N1 vector (Clontech) and the DOC-1R coding sequence was subcloned into pEGFP-C3 vector (Clontech) using a standard gene subcloning protocol. ..

    Reverse Transcription:

    Article Title: Involvement of the splicing factor SART1 in the BRCA1-dependent homologous recombination repair of DNA double-strand breaks
    Article Snippet: .. Human SART1 mRNA (NM_005146) was reverse-transcribed into complementary DNA (cDNA), which was cloned into the pEGFP-C3 vector (Clontech, Mountain View, CA, USA) using the In-Fusion HD cloning kit (Takara Bio, Shiga, Japan). .. SART1 mutants were generated using the KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan) with some modifications.

    Cloning:

    Article Title: Involvement of the splicing factor SART1 in the BRCA1-dependent homologous recombination repair of DNA double-strand breaks
    Article Snippet: .. Human SART1 mRNA (NM_005146) was reverse-transcribed into complementary DNA (cDNA), which was cloned into the pEGFP-C3 vector (Clontech, Mountain View, CA, USA) using the In-Fusion HD cloning kit (Takara Bio, Shiga, Japan). .. SART1 mutants were generated using the KOD-Plus-Mutagenesis kit (Toyobo, Osaka, Japan) with some modifications.

    Polymerase Chain Reaction:

    Article Title: RNAi-Mediated c-Rel Silencing Leads to Apoptosis of B Cell Tumor Cells and Suppresses Antigenic Immune Response In Vivo
    Article Snippet: .. The PCR product was cloned into the Bgl II and EcoR I sites of pEGFP-C3 vector (Clontech) to generate a new vector, pEGFP-mU6-1 , . ..

    Article Title: Biased exonization of transposed elements in duplicated genes: A lesson from the TIF-IA gene
    Article Snippet: .. A genomic DNA from 293T cell line (Gentra) corresponding to exon 1 through exon 2 of the TIF-IA gene (from locus 15) was PCR amplified and cloned into pEGFP-C3 vector (Clontech) between XhoI and BamHI sites under the control of the human cytomegalovirus (CMV) immediate early promoter, giving a ~1.9 kb insert. ..

    other:


    Sequencing:

    Article Title: Microbial metabolite n-butyrate upregulates intestinal claudin-23 expression through SP1 and AMPK pathways in mouse colon and human intestinal Caco-2 cells.
    Article Snippet: Aims: Regulation of the intestinal barrier is closely related to intestinal microbial metabolism.. This study investigated the role of intestinal microflora in the regulation of the tight junction (TJ) barrier in epithelial cells, focusing on the microbial metabolite n-butyrate, a major short-chain fatty acid, using mice and human intestinal

    Article Title: Overexpression of DOC-1R Inhibits Cell Cycle G1/S Transition by Repressing CDK2 Expression and Activation
    Article Snippet: For generation of pGST-DOC-1R and its truncated mutants, DOC-1R cDNA was amplified and subcloned into a pGEX-5X-1 vector (GE Healthcare, Piscataway, NJ). .. In addition, the CDK2 coding sequence was subcloned into a pDsRED-N1 vector (Clontech) and the DOC-1R coding sequence was subcloned into pEGFP-C3 vector (Clontech) using a standard gene subcloning protocol. ..

    Amplification:

    Article Title: STING guides the STX17-SNAP29-VAMP8 complex assembly to control autophagy
    Article Snippet: .. The STING cDNA was amplified and inserted into the p3xFLAG-Myc-CMV24 vector (Sigma) and pEGFP-C3 vector (Clontech) by HindIII and SalI to generate FLAG-STING and GFP-STING plasmids. ..

    Article Title: Biased exonization of transposed elements in duplicated genes: A lesson from the TIF-IA gene
    Article Snippet: .. A genomic DNA from 293T cell line (Gentra) corresponding to exon 1 through exon 2 of the TIF-IA gene (from locus 15) was PCR amplified and cloned into pEGFP-C3 vector (Clontech) between XhoI and BamHI sites under the control of the human cytomegalovirus (CMV) immediate early promoter, giving a ~1.9 kb insert. ..

    Control:

    Article Title: Biased exonization of transposed elements in duplicated genes: A lesson from the TIF-IA gene
    Article Snippet: .. A genomic DNA from 293T cell line (Gentra) corresponding to exon 1 through exon 2 of the TIF-IA gene (from locus 15) was PCR amplified and cloned into pEGFP-C3 vector (Clontech) between XhoI and BamHI sites under the control of the human cytomegalovirus (CMV) immediate early promoter, giving a ~1.9 kb insert. ..

    Subcloning:

    Article Title: Overexpression of DOC-1R Inhibits Cell Cycle G1/S Transition by Repressing CDK2 Expression and Activation
    Article Snippet: For generation of pGST-DOC-1R and its truncated mutants, DOC-1R cDNA was amplified and subcloned into a pGEX-5X-1 vector (GE Healthcare, Piscataway, NJ). .. In addition, the CDK2 coding sequence was subcloned into a pDsRED-N1 vector (Clontech) and the DOC-1R coding sequence was subcloned into pEGFP-C3 vector (Clontech) using a standard gene subcloning protocol. ..



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    a, Volcano plot of METTL3-interacting proteins in the cytoplasm of MCF7 cells. P < 0.12 and log 2 (METTL3/IgG) > 1 are used as thresholds. IgG is used as the negative control. The percentage of input used is 10%. b,c, Immunoprecipitation (IP) of cytoplasmic extracts from MDA-MB-231 with antibodies against METTL3 and <t>EXOC7</t> ( b ) and METTL14 ( c ) followed by immunoblotting with METTL3, EXOC7 and METTL14. IgG is used as the negative control. The percentage of input used is 10%. d, Immunoprecipitation of cytoplasmic extracts from MDA-MB-231 pretreated with RNase A with antibodies against METTL3 and EXOC7 followed by immunoblotting with METTL3, EXOC7 an METTL14. IgG is used as the negative control. Percentage of input is 10%. e, Schematic representation of GST–METTL3 purified proteins for in vitro pull-down analysis: GST–METTL3 full-length (FL), GST–N-terminal domain (NT), GST–zinc-finger domains (ZF), GST–leader-helix domain (LH) and GST–methylase-domain (MT). f, GST pull-down assay with in vitro transcribed and translated MYC–EXOC7. Immunoblotting of MYC–EXOC7 was detected using Streptavidin–HRP. GST-tagged proteins were detected with Ponceau red staining. GST alone was used as the negative control. The percentage of input used for the MYC–EXOC proteins is 1% and 10% for the GST-tagged proteins. Images are representative of three independently performed experiments with similar results.
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    a, Volcano plot of METTL3-interacting proteins in the cytoplasm of MCF7 cells. P < 0.12 and log 2 (METTL3/IgG) > 1 are used as thresholds. IgG is used as the negative control. The percentage of input used is 10%. b,c, Immunoprecipitation (IP) of cytoplasmic extracts from MDA-MB-231 with antibodies against METTL3 and <t>EXOC7</t> ( b ) and METTL14 ( c ) followed by immunoblotting with METTL3, EXOC7 and METTL14. IgG is used as the negative control. The percentage of input used is 10%. d, Immunoprecipitation of cytoplasmic extracts from MDA-MB-231 pretreated with RNase A with antibodies against METTL3 and EXOC7 followed by immunoblotting with METTL3, EXOC7 an METTL14. IgG is used as the negative control. Percentage of input is 10%. e, Schematic representation of GST–METTL3 purified proteins for in vitro pull-down analysis: GST–METTL3 full-length (FL), GST–N-terminal domain (NT), GST–zinc-finger domains (ZF), GST–leader-helix domain (LH) and GST–methylase-domain (MT). f, GST pull-down assay with in vitro transcribed and translated MYC–EXOC7. Immunoblotting of MYC–EXOC7 was detected using Streptavidin–HRP. GST-tagged proteins were detected with Ponceau red staining. GST alone was used as the negative control. The percentage of input used for the MYC–EXOC proteins is 1% and 10% for the GST-tagged proteins. Images are representative of three independently performed experiments with similar results.
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    Image Search Results


    a, Volcano plot of METTL3-interacting proteins in the cytoplasm of MCF7 cells. P < 0.12 and log 2 (METTL3/IgG) > 1 are used as thresholds. IgG is used as the negative control. The percentage of input used is 10%. b,c, Immunoprecipitation (IP) of cytoplasmic extracts from MDA-MB-231 with antibodies against METTL3 and EXOC7 ( b ) and METTL14 ( c ) followed by immunoblotting with METTL3, EXOC7 and METTL14. IgG is used as the negative control. The percentage of input used is 10%. d, Immunoprecipitation of cytoplasmic extracts from MDA-MB-231 pretreated with RNase A with antibodies against METTL3 and EXOC7 followed by immunoblotting with METTL3, EXOC7 an METTL14. IgG is used as the negative control. Percentage of input is 10%. e, Schematic representation of GST–METTL3 purified proteins for in vitro pull-down analysis: GST–METTL3 full-length (FL), GST–N-terminal domain (NT), GST–zinc-finger domains (ZF), GST–leader-helix domain (LH) and GST–methylase-domain (MT). f, GST pull-down assay with in vitro transcribed and translated MYC–EXOC7. Immunoblotting of MYC–EXOC7 was detected using Streptavidin–HRP. GST-tagged proteins were detected with Ponceau red staining. GST alone was used as the negative control. The percentage of input used for the MYC–EXOC proteins is 1% and 10% for the GST-tagged proteins. Images are representative of three independently performed experiments with similar results.

    Journal: bioRxiv

    Article Title: METTL3 regulates exocytosis independently of m 6 A

    doi: 10.1101/2025.05.26.656168

    Figure Lengend Snippet: a, Volcano plot of METTL3-interacting proteins in the cytoplasm of MCF7 cells. P < 0.12 and log 2 (METTL3/IgG) > 1 are used as thresholds. IgG is used as the negative control. The percentage of input used is 10%. b,c, Immunoprecipitation (IP) of cytoplasmic extracts from MDA-MB-231 with antibodies against METTL3 and EXOC7 ( b ) and METTL14 ( c ) followed by immunoblotting with METTL3, EXOC7 and METTL14. IgG is used as the negative control. The percentage of input used is 10%. d, Immunoprecipitation of cytoplasmic extracts from MDA-MB-231 pretreated with RNase A with antibodies against METTL3 and EXOC7 followed by immunoblotting with METTL3, EXOC7 an METTL14. IgG is used as the negative control. Percentage of input is 10%. e, Schematic representation of GST–METTL3 purified proteins for in vitro pull-down analysis: GST–METTL3 full-length (FL), GST–N-terminal domain (NT), GST–zinc-finger domains (ZF), GST–leader-helix domain (LH) and GST–methylase-domain (MT). f, GST pull-down assay with in vitro transcribed and translated MYC–EXOC7. Immunoblotting of MYC–EXOC7 was detected using Streptavidin–HRP. GST-tagged proteins were detected with Ponceau red staining. GST alone was used as the negative control. The percentage of input used for the MYC–EXOC proteins is 1% and 10% for the GST-tagged proteins. Images are representative of three independently performed experiments with similar results.

    Article Snippet: For EXOC7 overexpression and TNT formation assay, EXOC7 short (isoform 2) and long (isoform 6) isoforms were cloned into pEGFP-C3-EXO70 vector (Addgene #53761).

    Techniques: Negative Control, Immunoprecipitation, Western Blot, Purification, In Vitro, Pull Down Assay, Staining

    a, Quantitative PCR with reverse transcription (RT–qPCR) analysis of EXOC7 isoforms in scramble (scr) and METTL3-knockdown (sh1, sh2) cells. b, RT–PCR showing exon-skipping event in EXOC7 transcript in scramble (scr) and METTL3-knockdown (sh1, sh2) cells. Delta Percent Spliced-In (dPSI%) represents the change in exon inclusion levels between conditions. c, RT–PCR showing exon-skipping event in EXOC7 transcript in DMSO (control)- and STM2457-treated cells at indicated concentrations. dPSI% is indicated. d, Western blot of METTL3 and EXOC7 from whole-cell extracts from scramble (scr) and METTL3-knockdown (sh1, sh2) MDA-MB-231 cells showing EXOC7 protein isoforms. e, Western blot of METTL3 and EXOC7 from whole cell extracts from DMSO (control) and STM2457 treated cells at indicated concentrations. f, Western blots of EXOC7 from scramble (scr) and METTL3-knockdown (sh1, sh2) HEK293T cells during a 48 h CHX time course treatment. RPL22 is used as the loading control. g, Protein degradation curve of EXOC7 long isoform (6) during 48 h of CHX treatment generated from ( f ). P values at 24 h: METTL3-sh1 = 0.0292; METTL3-sh2 = 0.0363. Protein expression is normalized to RPL22 and relative to time 0 h. h, Protein degradation curve of EXOC7 short isoform (2) during 48 h of CHX treatment generated from ( f ). P values at 24 h: METTL3-sh1 = 0.0495; METTL3-sh2 = 0.2470. Protein expression is normalized to RPL22 and relative to time 0 h. i, Western blots of EXOC7 from DMSO-(control) and STM2457-treated HEK293T cells during a 48 h CHX time course treatment. RPL22 is used as the loading control. j, Protein degradation curve of EXOC7 long isoform (6) during 48 h of CHX treatment generated from ( i ). Protein expression is normalized to RPL22 and relative to time 0 h. k, Protein degradation curve of EXOC7 short isoform (2) during 48 h of CHX treatment generated from ( i ). Protein expression is normalized to RPL22 and relative to time 0 h. l, Non-reducing PAGE of EXOC7 from in vitro transcribed and translated Myc-EXOC7, scramble (scr) and METTL3-knockdown (sh1, sh2) MDA-MB-231 cells. β-Actin is used as the loading control. m, Western blot of EXOC7 from scramble (scr) and METTL3-knockdown (sh1, sh2) MDA-MB-231. β-Actin is used as the loading control. n, Non-reducing PAGE of EXOC7 from in vitro transcribed and translated Myc-EXOC7, DMSO (control)- and STM2457-treated MDA-MB-21 cells at indicated concentrations. β-Actin is used as the loading control. o, Western blot of EXOC7 from whole cell extracts from DMSO (control) and STM2457 treated cells at indicated concentrations. β-Actin is used as the loading control.

    Journal: bioRxiv

    Article Title: METTL3 regulates exocytosis independently of m 6 A

    doi: 10.1101/2025.05.26.656168

    Figure Lengend Snippet: a, Quantitative PCR with reverse transcription (RT–qPCR) analysis of EXOC7 isoforms in scramble (scr) and METTL3-knockdown (sh1, sh2) cells. b, RT–PCR showing exon-skipping event in EXOC7 transcript in scramble (scr) and METTL3-knockdown (sh1, sh2) cells. Delta Percent Spliced-In (dPSI%) represents the change in exon inclusion levels between conditions. c, RT–PCR showing exon-skipping event in EXOC7 transcript in DMSO (control)- and STM2457-treated cells at indicated concentrations. dPSI% is indicated. d, Western blot of METTL3 and EXOC7 from whole-cell extracts from scramble (scr) and METTL3-knockdown (sh1, sh2) MDA-MB-231 cells showing EXOC7 protein isoforms. e, Western blot of METTL3 and EXOC7 from whole cell extracts from DMSO (control) and STM2457 treated cells at indicated concentrations. f, Western blots of EXOC7 from scramble (scr) and METTL3-knockdown (sh1, sh2) HEK293T cells during a 48 h CHX time course treatment. RPL22 is used as the loading control. g, Protein degradation curve of EXOC7 long isoform (6) during 48 h of CHX treatment generated from ( f ). P values at 24 h: METTL3-sh1 = 0.0292; METTL3-sh2 = 0.0363. Protein expression is normalized to RPL22 and relative to time 0 h. h, Protein degradation curve of EXOC7 short isoform (2) during 48 h of CHX treatment generated from ( f ). P values at 24 h: METTL3-sh1 = 0.0495; METTL3-sh2 = 0.2470. Protein expression is normalized to RPL22 and relative to time 0 h. i, Western blots of EXOC7 from DMSO-(control) and STM2457-treated HEK293T cells during a 48 h CHX time course treatment. RPL22 is used as the loading control. j, Protein degradation curve of EXOC7 long isoform (6) during 48 h of CHX treatment generated from ( i ). Protein expression is normalized to RPL22 and relative to time 0 h. k, Protein degradation curve of EXOC7 short isoform (2) during 48 h of CHX treatment generated from ( i ). Protein expression is normalized to RPL22 and relative to time 0 h. l, Non-reducing PAGE of EXOC7 from in vitro transcribed and translated Myc-EXOC7, scramble (scr) and METTL3-knockdown (sh1, sh2) MDA-MB-231 cells. β-Actin is used as the loading control. m, Western blot of EXOC7 from scramble (scr) and METTL3-knockdown (sh1, sh2) MDA-MB-231. β-Actin is used as the loading control. n, Non-reducing PAGE of EXOC7 from in vitro transcribed and translated Myc-EXOC7, DMSO (control)- and STM2457-treated MDA-MB-21 cells at indicated concentrations. β-Actin is used as the loading control. o, Western blot of EXOC7 from whole cell extracts from DMSO (control) and STM2457 treated cells at indicated concentrations. β-Actin is used as the loading control.

    Article Snippet: For EXOC7 overexpression and TNT formation assay, EXOC7 short (isoform 2) and long (isoform 6) isoforms were cloned into pEGFP-C3-EXO70 vector (Addgene #53761).

    Techniques: Real-time Polymerase Chain Reaction, Reverse Transcription, Quantitative RT-PCR, Knockdown, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Generated, Expressing, In Vitro

    a, Left, immunofluorescence images of control (scr) and METTL3-knockdown (sh1 and sh2) HEK293T cells expressing GFP-VSV-Gts045 and collected after 3 h at 32 °C, then stained with the 8G5 antibody to visualize VSV-G incorporation into the plasma membrane. Scale bars, 30 µm. Right, quantification shows the ratio of extracellular (8G5 staining) to total cellular VSV-G fluorescence. b, Left, immunofluorescence images of HEK293T cells expressing GFP-VSV-Gts045 treated with DMSO (control) or STM2457 inhibitor after 3 h at 32 °C. Scale bars, 30 µm. Right, quantification shows the ratio of extracellular (8G5 staining) to total cellular VSV-G fluorescence. c–e, Correlation of secretome profiles between METTL3 and EXOC7 knockdowns ( c ), METTL3 knockdowns and STM2457-treated cells ( d ), and EXOC7 knockdowns and STM2457-treated cells ( e ). Proteins in common between different conditions were used to generate the plots. f, Overlap between upsecreted proteins from METTL3-knockdown (sh1 and sh2) and STM2457 treated MDA-MB-231 cells (log 2 fold change (FC) < 0 and false discovery rate (FDR) < 0.05). g, Overlap between proteins that exhibit downregulated secretion (downsecreted proteins) in METTL3-knockdown (sh1 and sh2) compared with STM2457 treated MDA-MB-231 cells (log 2 FC < 0 and FDR < 0.05). h, Overlap between downsecreted non-m 6 A-dependent proteins identified in g and upon EXOC7 knockdown (sh1 and sh2) (log 2 FC < 0 and FDR < 0.05). i, Gene ontology (GO) analysis of biological processes related to the common downsecreted proteins identified in h . j , Western blot of METTL3, intracellular and extracellular MMP1 from scramble (scr) and METTL3-knockdown (sh1, sh2) MDA-MB-231 cells showing lower secretion of MMP1 compared to intracellular levels. k , Western blot of intracellular and extracellular MMP1 from DMSO- and STM2457-treated cells showing no defect in MMP1 secretion compared to intracellular levels upon METTL3 inhibition. Data are mean ± s.d. One-way ANOVA with Dunnett’s correction for multiple comparison ( a ) and two-tailed Student’s t-test ( b ).

    Journal: bioRxiv

    Article Title: METTL3 regulates exocytosis independently of m 6 A

    doi: 10.1101/2025.05.26.656168

    Figure Lengend Snippet: a, Left, immunofluorescence images of control (scr) and METTL3-knockdown (sh1 and sh2) HEK293T cells expressing GFP-VSV-Gts045 and collected after 3 h at 32 °C, then stained with the 8G5 antibody to visualize VSV-G incorporation into the plasma membrane. Scale bars, 30 µm. Right, quantification shows the ratio of extracellular (8G5 staining) to total cellular VSV-G fluorescence. b, Left, immunofluorescence images of HEK293T cells expressing GFP-VSV-Gts045 treated with DMSO (control) or STM2457 inhibitor after 3 h at 32 °C. Scale bars, 30 µm. Right, quantification shows the ratio of extracellular (8G5 staining) to total cellular VSV-G fluorescence. c–e, Correlation of secretome profiles between METTL3 and EXOC7 knockdowns ( c ), METTL3 knockdowns and STM2457-treated cells ( d ), and EXOC7 knockdowns and STM2457-treated cells ( e ). Proteins in common between different conditions were used to generate the plots. f, Overlap between upsecreted proteins from METTL3-knockdown (sh1 and sh2) and STM2457 treated MDA-MB-231 cells (log 2 fold change (FC) < 0 and false discovery rate (FDR) < 0.05). g, Overlap between proteins that exhibit downregulated secretion (downsecreted proteins) in METTL3-knockdown (sh1 and sh2) compared with STM2457 treated MDA-MB-231 cells (log 2 FC < 0 and FDR < 0.05). h, Overlap between downsecreted non-m 6 A-dependent proteins identified in g and upon EXOC7 knockdown (sh1 and sh2) (log 2 FC < 0 and FDR < 0.05). i, Gene ontology (GO) analysis of biological processes related to the common downsecreted proteins identified in h . j , Western blot of METTL3, intracellular and extracellular MMP1 from scramble (scr) and METTL3-knockdown (sh1, sh2) MDA-MB-231 cells showing lower secretion of MMP1 compared to intracellular levels. k , Western blot of intracellular and extracellular MMP1 from DMSO- and STM2457-treated cells showing no defect in MMP1 secretion compared to intracellular levels upon METTL3 inhibition. Data are mean ± s.d. One-way ANOVA with Dunnett’s correction for multiple comparison ( a ) and two-tailed Student’s t-test ( b ).

    Article Snippet: For EXOC7 overexpression and TNT formation assay, EXOC7 short (isoform 2) and long (isoform 6) isoforms were cloned into pEGFP-C3-EXO70 vector (Addgene #53761).

    Techniques: Immunofluorescence, Control, Knockdown, Expressing, Staining, Clinical Proteomics, Membrane, Fluorescence, Western Blot, Inhibition, Comparison, Two Tailed Test