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mruby2 n1  (Addgene inc)


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    Structured Review

    Addgene inc mruby2 n1
    Mruby2 N1, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mruby2+n1/mRuby2-N1+(Plasmid+%2354614)/us12351877-394-25-31
    Average 93 stars, based on 18 article reviews
    mruby2 n1 - by Bioz Stars, 2026-11
    93/100 stars

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    Related Articles

    Construct:

    Article Title: Systems and methods for determining a treatment course of action
    Article Snippet: .. Lentiviral construct pLenti CMV-MRLC1-mRuby2-IRES-PuroR was generated by PCR amplification of cDNA for Homo sapiens MRLC1 from eGFP-MRLC (a gift from Tom Egelhoff, Addgene #35680) and mRuby2-N1 (a gift from Michael Davidson, Addgene #54614) to create C-terminal tagged MRLC. .. Fusion construct was subcloned into the expression vector pCIG3 (pCMVIRES-GFP, a gift from Felicia Goodrum, Addgene plasmid #78264), modified to replace the GFP cassette with puromycin resistance gene. pCMV-iRFP670-EVL-IRES-BlastR was generated by PCR amplification of cDNA for Mus musculus EVL cDNA from MSCV-eGFP-EVL and piRFP670-N1 (a gift from Vladislav Verkhusha, Addgene #45457) to create N-terminal tagged EVL.

    Article Title: IL-24 intrinsically regulates Th17 cell pathogenicity in mice
    Article Snippet: The sequences corresponding to full-length or nonsecretable (truncated) versions of IL-24, i.e., IL-24 and IL-24t, were cloned into mammalian expression vector pIRES-eGFP (6029-1; Clontech). .. The fusion constructs of nonsecretable IL-24 (mRuby2-N1-IL-24t; sequence: 5′-ATG CAA GAG TTC CGA TTT GGG TCT TGC CAA GTG ACA GGG GTG GTT CTC CCA GAA CTG TGG GAG GCC TTC TGG ACT GTG AAG AAC ACT GTG CAA ACT CAG GAT GAC ATC ACA AGC ATC CGG CTG TTG AAG CCG CAG GTT CTG CGG AAT GTC TCG GGT GCT GAG AGC TGT TAC CTT GCC CAC AGC CTG CTG AAG TTC TAC TTG AAC ACT GTT TTC AAG AAC TAC CAC AGC AAA ATA GCC AAA TTC AAG GTC TTG AGG TCA TTC TCC ACT CTG GCC AAC AAC TTC ATA GTC ATC ATG TCA CAA CTA CAG CCC AGT AAG GAC AAT TCC ATG CTT CCC ATT AGT GAG AGT GCA CAC CAG CGG TTT TTG CTG TTC CGC AGA GCA TTC AAA CAG TTG GAT ACA GAA GTC GCT TTG GTG AAA GCC TTT GGG GAA GTG GAC ATT CTC CTG ACC TGG ATG CAG AAA TTC TAC CAT CTC-3′) or IFN-γ (mRuby2-N1-IFN-γt; sequence: 5′-ATG CAC GGC ACA GTC ATT GAA AGC CTA GAA AGT CTG AAT AAC TAT TTT AAC TCA AGT GGC ATA GAT GTG GAA GAA AAG AGT CTC TTC TTG GAT ATC TGG AGG AAC TGG CAA AAG GAT GGT GAC ATG AAA ATC CTG CAG AGC CAG ATT ATC TCT TTC TAC CTC AGA CTC TTT GAA GTC TTG AAA GAC AAT CAG GCC ATC AGC AAC AAC ATA AGC GTC ATT GAA TCA CAC CTG ATT ACT ACC TTC TTC AGC AAC AGC AAG GCG AAA AAG GAT GCA TTC ATG AGT ATT GCC AAG TTT GAG GTC AAC AAC CCA CAG GTC CAG CGC CAA GCA TTC AAT GAG CTC ATC CGA GTG GTC CAC CAG CTG TTG CCG GAA TCC AGC CTC AGG AAG CGG AAA AGG AGT CGC TGC-3′) were generated by subcloning into mRuby2-N1 (54614; Addgene). .. The gRNAs targeting IL-24 or GFP were cloned into a retroviral vector (pMSCV-U6-guide-IRES-Thy1.1) using Gibson’s assembly kit (E2611S; New England Biolabs).

    Generated:

    Article Title: Systems and methods for determining a treatment course of action
    Article Snippet: .. Lentiviral construct pLenti CMV-MRLC1-mRuby2-IRES-PuroR was generated by PCR amplification of cDNA for Homo sapiens MRLC1 from eGFP-MRLC (a gift from Tom Egelhoff, Addgene #35680) and mRuby2-N1 (a gift from Michael Davidson, Addgene #54614) to create C-terminal tagged MRLC. .. Fusion construct was subcloned into the expression vector pCIG3 (pCMVIRES-GFP, a gift from Felicia Goodrum, Addgene plasmid #78264), modified to replace the GFP cassette with puromycin resistance gene. pCMV-iRFP670-EVL-IRES-BlastR was generated by PCR amplification of cDNA for Mus musculus EVL cDNA from MSCV-eGFP-EVL and piRFP670-N1 (a gift from Vladislav Verkhusha, Addgene #45457) to create N-terminal tagged EVL.

    Article Title: IL-24 intrinsically regulates Th17 cell pathogenicity in mice
    Article Snippet: The sequences corresponding to full-length or nonsecretable (truncated) versions of IL-24, i.e., IL-24 and IL-24t, were cloned into mammalian expression vector pIRES-eGFP (6029-1; Clontech). .. The fusion constructs of nonsecretable IL-24 (mRuby2-N1-IL-24t; sequence: 5′-ATG CAA GAG TTC CGA TTT GGG TCT TGC CAA GTG ACA GGG GTG GTT CTC CCA GAA CTG TGG GAG GCC TTC TGG ACT GTG AAG AAC ACT GTG CAA ACT CAG GAT GAC ATC ACA AGC ATC CGG CTG TTG AAG CCG CAG GTT CTG CGG AAT GTC TCG GGT GCT GAG AGC TGT TAC CTT GCC CAC AGC CTG CTG AAG TTC TAC TTG AAC ACT GTT TTC AAG AAC TAC CAC AGC AAA ATA GCC AAA TTC AAG GTC TTG AGG TCA TTC TCC ACT CTG GCC AAC AAC TTC ATA GTC ATC ATG TCA CAA CTA CAG CCC AGT AAG GAC AAT TCC ATG CTT CCC ATT AGT GAG AGT GCA CAC CAG CGG TTT TTG CTG TTC CGC AGA GCA TTC AAA CAG TTG GAT ACA GAA GTC GCT TTG GTG AAA GCC TTT GGG GAA GTG GAC ATT CTC CTG ACC TGG ATG CAG AAA TTC TAC CAT CTC-3′) or IFN-γ (mRuby2-N1-IFN-γt; sequence: 5′-ATG CAC GGC ACA GTC ATT GAA AGC CTA GAA AGT CTG AAT AAC TAT TTT AAC TCA AGT GGC ATA GAT GTG GAA GAA AAG AGT CTC TTC TTG GAT ATC TGG AGG AAC TGG CAA AAG GAT GGT GAC ATG AAA ATC CTG CAG AGC CAG ATT ATC TCT TTC TAC CTC AGA CTC TTT GAA GTC TTG AAA GAC AAT CAG GCC ATC AGC AAC AAC ATA AGC GTC ATT GAA TCA CAC CTG ATT ACT ACC TTC TTC AGC AAC AGC AAG GCG AAA AAG GAT GCA TTC ATG AGT ATT GCC AAG TTT GAG GTC AAC AAC CCA CAG GTC CAG CGC CAA GCA TTC AAT GAG CTC ATC CGA GTG GTC CAC CAG CTG TTG CCG GAA TCC AGC CTC AGG AAG CGG AAA AGG AGT CGC TGC-3′) were generated by subcloning into mRuby2-N1 (54614; Addgene). .. The gRNAs targeting IL-24 or GFP were cloned into a retroviral vector (pMSCV-U6-guide-IRES-Thy1.1) using Gibson’s assembly kit (E2611S; New England Biolabs).

    Polymerase Chain Reaction:

    Article Title: Systems and methods for determining a treatment course of action
    Article Snippet: .. Lentiviral construct pLenti CMV-MRLC1-mRuby2-IRES-PuroR was generated by PCR amplification of cDNA for Homo sapiens MRLC1 from eGFP-MRLC (a gift from Tom Egelhoff, Addgene #35680) and mRuby2-N1 (a gift from Michael Davidson, Addgene #54614) to create C-terminal tagged MRLC. .. Fusion construct was subcloned into the expression vector pCIG3 (pCMVIRES-GFP, a gift from Felicia Goodrum, Addgene plasmid #78264), modified to replace the GFP cassette with puromycin resistance gene. pCMV-iRFP670-EVL-IRES-BlastR was generated by PCR amplification of cDNA for Mus musculus EVL cDNA from MSCV-eGFP-EVL and piRFP670-N1 (a gift from Vladislav Verkhusha, Addgene #45457) to create N-terminal tagged EVL.

    Amplification:

    Article Title: Systems and methods for determining a treatment course of action
    Article Snippet: .. Lentiviral construct pLenti CMV-MRLC1-mRuby2-IRES-PuroR was generated by PCR amplification of cDNA for Homo sapiens MRLC1 from eGFP-MRLC (a gift from Tom Egelhoff, Addgene #35680) and mRuby2-N1 (a gift from Michael Davidson, Addgene #54614) to create C-terminal tagged MRLC. .. Fusion construct was subcloned into the expression vector pCIG3 (pCMVIRES-GFP, a gift from Felicia Goodrum, Addgene plasmid #78264), modified to replace the GFP cassette with puromycin resistance gene. pCMV-iRFP670-EVL-IRES-BlastR was generated by PCR amplification of cDNA for Mus musculus EVL cDNA from MSCV-eGFP-EVL and piRFP670-N1 (a gift from Vladislav Verkhusha, Addgene #45457) to create N-terminal tagged EVL.

    Article Title: Hijacking of transcriptional condensates by endogenous retroviruses.
    Article Snippet: To knock in the degradation-sensitive FKBPF36V tag at the N-terminus of TRIM28, a repair template containing homology arms spanning upstream and downstream of the target site was cloned into a pUC19 vector (NEB) (Supplementary Fig. 2a). .. The repair template included a mRuby2 fluorescent protein sequence, P2A linker and the FKBP tag sequence (Supplementary Fig. 2a)25. mRuby2 sequence was amplified from the mRuby2-N1 plasmid (Addgene, catalog no. 54614), and the P2A-FKBP sequence was amplified from the PITCh dTAG donor vector (Addgene, catalog no. 91792). .. A guide RNA (Supplementary Table 1b) targeting the N-terminus of TRIM28 was cloned into the sgRNA-Cas9 vector pX458 (Addgene, catalog no. 48138).

    Sequencing:

    Article Title: IL-24 intrinsically regulates Th17 cell pathogenicity in mice
    Article Snippet: The sequences corresponding to full-length or nonsecretable (truncated) versions of IL-24, i.e., IL-24 and IL-24t, were cloned into mammalian expression vector pIRES-eGFP (6029-1; Clontech). .. The fusion constructs of nonsecretable IL-24 (mRuby2-N1-IL-24t; sequence: 5′-ATG CAA GAG TTC CGA TTT GGG TCT TGC CAA GTG ACA GGG GTG GTT CTC CCA GAA CTG TGG GAG GCC TTC TGG ACT GTG AAG AAC ACT GTG CAA ACT CAG GAT GAC ATC ACA AGC ATC CGG CTG TTG AAG CCG CAG GTT CTG CGG AAT GTC TCG GGT GCT GAG AGC TGT TAC CTT GCC CAC AGC CTG CTG AAG TTC TAC TTG AAC ACT GTT TTC AAG AAC TAC CAC AGC AAA ATA GCC AAA TTC AAG GTC TTG AGG TCA TTC TCC ACT CTG GCC AAC AAC TTC ATA GTC ATC ATG TCA CAA CTA CAG CCC AGT AAG GAC AAT TCC ATG CTT CCC ATT AGT GAG AGT GCA CAC CAG CGG TTT TTG CTG TTC CGC AGA GCA TTC AAA CAG TTG GAT ACA GAA GTC GCT TTG GTG AAA GCC TTT GGG GAA GTG GAC ATT CTC CTG ACC TGG ATG CAG AAA TTC TAC CAT CTC-3′) or IFN-γ (mRuby2-N1-IFN-γt; sequence: 5′-ATG CAC GGC ACA GTC ATT GAA AGC CTA GAA AGT CTG AAT AAC TAT TTT AAC TCA AGT GGC ATA GAT GTG GAA GAA AAG AGT CTC TTC TTG GAT ATC TGG AGG AAC TGG CAA AAG GAT GGT GAC ATG AAA ATC CTG CAG AGC CAG ATT ATC TCT TTC TAC CTC AGA CTC TTT GAA GTC TTG AAA GAC AAT CAG GCC ATC AGC AAC AAC ATA AGC GTC ATT GAA TCA CAC CTG ATT ACT ACC TTC TTC AGC AAC AGC AAG GCG AAA AAG GAT GCA TTC ATG AGT ATT GCC AAG TTT GAG GTC AAC AAC CCA CAG GTC CAG CGC CAA GCA TTC AAT GAG CTC ATC CGA GTG GTC CAC CAG CTG TTG CCG GAA TCC AGC CTC AGG AAG CGG AAA AGG AGT CGC TGC-3′) were generated by subcloning into mRuby2-N1 (54614; Addgene). .. The gRNAs targeting IL-24 or GFP were cloned into a retroviral vector (pMSCV-U6-guide-IRES-Thy1.1) using Gibson’s assembly kit (E2611S; New England Biolabs).

    Article Title: Hijacking of transcriptional condensates by endogenous retroviruses.
    Article Snippet: To knock in the degradation-sensitive FKBPF36V tag at the N-terminus of TRIM28, a repair template containing homology arms spanning upstream and downstream of the target site was cloned into a pUC19 vector (NEB) (Supplementary Fig. 2a). .. The repair template included a mRuby2 fluorescent protein sequence, P2A linker and the FKBP tag sequence (Supplementary Fig. 2a)25. mRuby2 sequence was amplified from the mRuby2-N1 plasmid (Addgene, catalog no. 54614), and the P2A-FKBP sequence was amplified from the PITCh dTAG donor vector (Addgene, catalog no. 91792). .. A guide RNA (Supplementary Table 1b) targeting the N-terminus of TRIM28 was cloned into the sgRNA-Cas9 vector pX458 (Addgene, catalog no. 48138).

    Cellular Antioxidant Activity Assay:

    Article Title: IL-24 intrinsically regulates Th17 cell pathogenicity in mice
    Article Snippet: The sequences corresponding to full-length or nonsecretable (truncated) versions of IL-24, i.e., IL-24 and IL-24t, were cloned into mammalian expression vector pIRES-eGFP (6029-1; Clontech). .. The fusion constructs of nonsecretable IL-24 (mRuby2-N1-IL-24t; sequence: 5′-ATG CAA GAG TTC CGA TTT GGG TCT TGC CAA GTG ACA GGG GTG GTT CTC CCA GAA CTG TGG GAG GCC TTC TGG ACT GTG AAG AAC ACT GTG CAA ACT CAG GAT GAC ATC ACA AGC ATC CGG CTG TTG AAG CCG CAG GTT CTG CGG AAT GTC TCG GGT GCT GAG AGC TGT TAC CTT GCC CAC AGC CTG CTG AAG TTC TAC TTG AAC ACT GTT TTC AAG AAC TAC CAC AGC AAA ATA GCC AAA TTC AAG GTC TTG AGG TCA TTC TCC ACT CTG GCC AAC AAC TTC ATA GTC ATC ATG TCA CAA CTA CAG CCC AGT AAG GAC AAT TCC ATG CTT CCC ATT AGT GAG AGT GCA CAC CAG CGG TTT TTG CTG TTC CGC AGA GCA TTC AAA CAG TTG GAT ACA GAA GTC GCT TTG GTG AAA GCC TTT GGG GAA GTG GAC ATT CTC CTG ACC TGG ATG CAG AAA TTC TAC CAT CTC-3′) or IFN-γ (mRuby2-N1-IFN-γt; sequence: 5′-ATG CAC GGC ACA GTC ATT GAA AGC CTA GAA AGT CTG AAT AAC TAT TTT AAC TCA AGT GGC ATA GAT GTG GAA GAA AAG AGT CTC TTC TTG GAT ATC TGG AGG AAC TGG CAA AAG GAT GGT GAC ATG AAA ATC CTG CAG AGC CAG ATT ATC TCT TTC TAC CTC AGA CTC TTT GAA GTC TTG AAA GAC AAT CAG GCC ATC AGC AAC AAC ATA AGC GTC ATT GAA TCA CAC CTG ATT ACT ACC TTC TTC AGC AAC AGC AAG GCG AAA AAG GAT GCA TTC ATG AGT ATT GCC AAG TTT GAG GTC AAC AAC CCA CAG GTC CAG CGC CAA GCA TTC AAT GAG CTC ATC CGA GTG GTC CAC CAG CTG TTG CCG GAA TCC AGC CTC AGG AAG CGG AAA AGG AGT CGC TGC-3′) were generated by subcloning into mRuby2-N1 (54614; Addgene). .. The gRNAs targeting IL-24 or GFP were cloned into a retroviral vector (pMSCV-U6-guide-IRES-Thy1.1) using Gibson’s assembly kit (E2611S; New England Biolabs).

    CTG Assay:

    Article Title: IL-24 intrinsically regulates Th17 cell pathogenicity in mice
    Article Snippet: The sequences corresponding to full-length or nonsecretable (truncated) versions of IL-24, i.e., IL-24 and IL-24t, were cloned into mammalian expression vector pIRES-eGFP (6029-1; Clontech). .. The fusion constructs of nonsecretable IL-24 (mRuby2-N1-IL-24t; sequence: 5′-ATG CAA GAG TTC CGA TTT GGG TCT TGC CAA GTG ACA GGG GTG GTT CTC CCA GAA CTG TGG GAG GCC TTC TGG ACT GTG AAG AAC ACT GTG CAA ACT CAG GAT GAC ATC ACA AGC ATC CGG CTG TTG AAG CCG CAG GTT CTG CGG AAT GTC TCG GGT GCT GAG AGC TGT TAC CTT GCC CAC AGC CTG CTG AAG TTC TAC TTG AAC ACT GTT TTC AAG AAC TAC CAC AGC AAA ATA GCC AAA TTC AAG GTC TTG AGG TCA TTC TCC ACT CTG GCC AAC AAC TTC ATA GTC ATC ATG TCA CAA CTA CAG CCC AGT AAG GAC AAT TCC ATG CTT CCC ATT AGT GAG AGT GCA CAC CAG CGG TTT TTG CTG TTC CGC AGA GCA TTC AAA CAG TTG GAT ACA GAA GTC GCT TTG GTG AAA GCC TTT GGG GAA GTG GAC ATT CTC CTG ACC TGG ATG CAG AAA TTC TAC CAT CTC-3′) or IFN-γ (mRuby2-N1-IFN-γt; sequence: 5′-ATG CAC GGC ACA GTC ATT GAA AGC CTA GAA AGT CTG AAT AAC TAT TTT AAC TCA AGT GGC ATA GAT GTG GAA GAA AAG AGT CTC TTC TTG GAT ATC TGG AGG AAC TGG CAA AAG GAT GGT GAC ATG AAA ATC CTG CAG AGC CAG ATT ATC TCT TTC TAC CTC AGA CTC TTT GAA GTC TTG AAA GAC AAT CAG GCC ATC AGC AAC AAC ATA AGC GTC ATT GAA TCA CAC CTG ATT ACT ACC TTC TTC AGC AAC AGC AAG GCG AAA AAG GAT GCA TTC ATG AGT ATT GCC AAG TTT GAG GTC AAC AAC CCA CAG GTC CAG CGC CAA GCA TTC AAT GAG CTC ATC CGA GTG GTC CAC CAG CTG TTG CCG GAA TCC AGC CTC AGG AAG CGG AAA AGG AGT CGC TGC-3′) were generated by subcloning into mRuby2-N1 (54614; Addgene). .. The gRNAs targeting IL-24 or GFP were cloned into a retroviral vector (pMSCV-U6-guide-IRES-Thy1.1) using Gibson’s assembly kit (E2611S; New England Biolabs).

    Activated Clotting Time Assay:

    Article Title: IL-24 intrinsically regulates Th17 cell pathogenicity in mice
    Article Snippet: The sequences corresponding to full-length or nonsecretable (truncated) versions of IL-24, i.e., IL-24 and IL-24t, were cloned into mammalian expression vector pIRES-eGFP (6029-1; Clontech). .. The fusion constructs of nonsecretable IL-24 (mRuby2-N1-IL-24t; sequence: 5′-ATG CAA GAG TTC CGA TTT GGG TCT TGC CAA GTG ACA GGG GTG GTT CTC CCA GAA CTG TGG GAG GCC TTC TGG ACT GTG AAG AAC ACT GTG CAA ACT CAG GAT GAC ATC ACA AGC ATC CGG CTG TTG AAG CCG CAG GTT CTG CGG AAT GTC TCG GGT GCT GAG AGC TGT TAC CTT GCC CAC AGC CTG CTG AAG TTC TAC TTG AAC ACT GTT TTC AAG AAC TAC CAC AGC AAA ATA GCC AAA TTC AAG GTC TTG AGG TCA TTC TCC ACT CTG GCC AAC AAC TTC ATA GTC ATC ATG TCA CAA CTA CAG CCC AGT AAG GAC AAT TCC ATG CTT CCC ATT AGT GAG AGT GCA CAC CAG CGG TTT TTG CTG TTC CGC AGA GCA TTC AAA CAG TTG GAT ACA GAA GTC GCT TTG GTG AAA GCC TTT GGG GAA GTG GAC ATT CTC CTG ACC TGG ATG CAG AAA TTC TAC CAT CTC-3′) or IFN-γ (mRuby2-N1-IFN-γt; sequence: 5′-ATG CAC GGC ACA GTC ATT GAA AGC CTA GAA AGT CTG AAT AAC TAT TTT AAC TCA AGT GGC ATA GAT GTG GAA GAA AAG AGT CTC TTC TTG GAT ATC TGG AGG AAC TGG CAA AAG GAT GGT GAC ATG AAA ATC CTG CAG AGC CAG ATT ATC TCT TTC TAC CTC AGA CTC TTT GAA GTC TTG AAA GAC AAT CAG GCC ATC AGC AAC AAC ATA AGC GTC ATT GAA TCA CAC CTG ATT ACT ACC TTC TTC AGC AAC AGC AAG GCG AAA AAG GAT GCA TTC ATG AGT ATT GCC AAG TTT GAG GTC AAC AAC CCA CAG GTC CAG CGC CAA GCA TTC AAT GAG CTC ATC CGA GTG GTC CAC CAG CTG TTG CCG GAA TCC AGC CTC AGG AAG CGG AAA AGG AGT CGC TGC-3′) were generated by subcloning into mRuby2-N1 (54614; Addgene). .. The gRNAs targeting IL-24 or GFP were cloned into a retroviral vector (pMSCV-U6-guide-IRES-Thy1.1) using Gibson’s assembly kit (E2611S; New England Biolabs).

    Countercurrent Chromatography:

    Article Title: IL-24 intrinsically regulates Th17 cell pathogenicity in mice
    Article Snippet: The sequences corresponding to full-length or nonsecretable (truncated) versions of IL-24, i.e., IL-24 and IL-24t, were cloned into mammalian expression vector pIRES-eGFP (6029-1; Clontech). .. The fusion constructs of nonsecretable IL-24 (mRuby2-N1-IL-24t; sequence: 5′-ATG CAA GAG TTC CGA TTT GGG TCT TGC CAA GTG ACA GGG GTG GTT CTC CCA GAA CTG TGG GAG GCC TTC TGG ACT GTG AAG AAC ACT GTG CAA ACT CAG GAT GAC ATC ACA AGC ATC CGG CTG TTG AAG CCG CAG GTT CTG CGG AAT GTC TCG GGT GCT GAG AGC TGT TAC CTT GCC CAC AGC CTG CTG AAG TTC TAC TTG AAC ACT GTT TTC AAG AAC TAC CAC AGC AAA ATA GCC AAA TTC AAG GTC TTG AGG TCA TTC TCC ACT CTG GCC AAC AAC TTC ATA GTC ATC ATG TCA CAA CTA CAG CCC AGT AAG GAC AAT TCC ATG CTT CCC ATT AGT GAG AGT GCA CAC CAG CGG TTT TTG CTG TTC CGC AGA GCA TTC AAA CAG TTG GAT ACA GAA GTC GCT TTG GTG AAA GCC TTT GGG GAA GTG GAC ATT CTC CTG ACC TGG ATG CAG AAA TTC TAC CAT CTC-3′) or IFN-γ (mRuby2-N1-IFN-γt; sequence: 5′-ATG CAC GGC ACA GTC ATT GAA AGC CTA GAA AGT CTG AAT AAC TAT TTT AAC TCA AGT GGC ATA GAT GTG GAA GAA AAG AGT CTC TTC TTG GAT ATC TGG AGG AAC TGG CAA AAG GAT GGT GAC ATG AAA ATC CTG CAG AGC CAG ATT ATC TCT TTC TAC CTC AGA CTC TTT GAA GTC TTG AAA GAC AAT CAG GCC ATC AGC AAC AAC ATA AGC GTC ATT GAA TCA CAC CTG ATT ACT ACC TTC TTC AGC AAC AGC AAG GCG AAA AAG GAT GCA TTC ATG AGT ATT GCC AAG TTT GAG GTC AAC AAC CCA CAG GTC CAG CGC CAA GCA TTC AAT GAG CTC ATC CGA GTG GTC CAC CAG CTG TTG CCG GAA TCC AGC CTC AGG AAG CGG AAA AGG AGT CGC TGC-3′) were generated by subcloning into mRuby2-N1 (54614; Addgene). .. The gRNAs targeting IL-24 or GFP were cloned into a retroviral vector (pMSCV-U6-guide-IRES-Thy1.1) using Gibson’s assembly kit (E2611S; New England Biolabs).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: IL-24 intrinsically regulates Th17 cell pathogenicity in mice
    Article Snippet: The sequences corresponding to full-length or nonsecretable (truncated) versions of IL-24, i.e., IL-24 and IL-24t, were cloned into mammalian expression vector pIRES-eGFP (6029-1; Clontech). .. The fusion constructs of nonsecretable IL-24 (mRuby2-N1-IL-24t; sequence: 5′-ATG CAA GAG TTC CGA TTT GGG TCT TGC CAA GTG ACA GGG GTG GTT CTC CCA GAA CTG TGG GAG GCC TTC TGG ACT GTG AAG AAC ACT GTG CAA ACT CAG GAT GAC ATC ACA AGC ATC CGG CTG TTG AAG CCG CAG GTT CTG CGG AAT GTC TCG GGT GCT GAG AGC TGT TAC CTT GCC CAC AGC CTG CTG AAG TTC TAC TTG AAC ACT GTT TTC AAG AAC TAC CAC AGC AAA ATA GCC AAA TTC AAG GTC TTG AGG TCA TTC TCC ACT CTG GCC AAC AAC TTC ATA GTC ATC ATG TCA CAA CTA CAG CCC AGT AAG GAC AAT TCC ATG CTT CCC ATT AGT GAG AGT GCA CAC CAG CGG TTT TTG CTG TTC CGC AGA GCA TTC AAA CAG TTG GAT ACA GAA GTC GCT TTG GTG AAA GCC TTT GGG GAA GTG GAC ATT CTC CTG ACC TGG ATG CAG AAA TTC TAC CAT CTC-3′) or IFN-γ (mRuby2-N1-IFN-γt; sequence: 5′-ATG CAC GGC ACA GTC ATT GAA AGC CTA GAA AGT CTG AAT AAC TAT TTT AAC TCA AGT GGC ATA GAT GTG GAA GAA AAG AGT CTC TTC TTG GAT ATC TGG AGG AAC TGG CAA AAG GAT GGT GAC ATG AAA ATC CTG CAG AGC CAG ATT ATC TCT TTC TAC CTC AGA CTC TTT GAA GTC TTG AAA GAC AAT CAG GCC ATC AGC AAC AAC ATA AGC GTC ATT GAA TCA CAC CTG ATT ACT ACC TTC TTC AGC AAC AGC AAG GCG AAA AAG GAT GCA TTC ATG AGT ATT GCC AAG TTT GAG GTC AAC AAC CCA CAG GTC CAG CGC CAA GCA TTC AAT GAG CTC ATC CGA GTG GTC CAC CAG CTG TTG CCG GAA TCC AGC CTC AGG AAG CGG AAA AGG AGT CGC TGC-3′) were generated by subcloning into mRuby2-N1 (54614; Addgene). .. The gRNAs targeting IL-24 or GFP were cloned into a retroviral vector (pMSCV-U6-guide-IRES-Thy1.1) using Gibson’s assembly kit (E2611S; New England Biolabs).

    Subcloning:

    Article Title: IL-24 intrinsically regulates Th17 cell pathogenicity in mice
    Article Snippet: The sequences corresponding to full-length or nonsecretable (truncated) versions of IL-24, i.e., IL-24 and IL-24t, were cloned into mammalian expression vector pIRES-eGFP (6029-1; Clontech). .. The fusion constructs of nonsecretable IL-24 (mRuby2-N1-IL-24t; sequence: 5′-ATG CAA GAG TTC CGA TTT GGG TCT TGC CAA GTG ACA GGG GTG GTT CTC CCA GAA CTG TGG GAG GCC TTC TGG ACT GTG AAG AAC ACT GTG CAA ACT CAG GAT GAC ATC ACA AGC ATC CGG CTG TTG AAG CCG CAG GTT CTG CGG AAT GTC TCG GGT GCT GAG AGC TGT TAC CTT GCC CAC AGC CTG CTG AAG TTC TAC TTG AAC ACT GTT TTC AAG AAC TAC CAC AGC AAA ATA GCC AAA TTC AAG GTC TTG AGG TCA TTC TCC ACT CTG GCC AAC AAC TTC ATA GTC ATC ATG TCA CAA CTA CAG CCC AGT AAG GAC AAT TCC ATG CTT CCC ATT AGT GAG AGT GCA CAC CAG CGG TTT TTG CTG TTC CGC AGA GCA TTC AAA CAG TTG GAT ACA GAA GTC GCT TTG GTG AAA GCC TTT GGG GAA GTG GAC ATT CTC CTG ACC TGG ATG CAG AAA TTC TAC CAT CTC-3′) or IFN-γ (mRuby2-N1-IFN-γt; sequence: 5′-ATG CAC GGC ACA GTC ATT GAA AGC CTA GAA AGT CTG AAT AAC TAT TTT AAC TCA AGT GGC ATA GAT GTG GAA GAA AAG AGT CTC TTC TTG GAT ATC TGG AGG AAC TGG CAA AAG GAT GGT GAC ATG AAA ATC CTG CAG AGC CAG ATT ATC TCT TTC TAC CTC AGA CTC TTT GAA GTC TTG AAA GAC AAT CAG GCC ATC AGC AAC AAC ATA AGC GTC ATT GAA TCA CAC CTG ATT ACT ACC TTC TTC AGC AAC AGC AAG GCG AAA AAG GAT GCA TTC ATG AGT ATT GCC AAG TTT GAG GTC AAC AAC CCA CAG GTC CAG CGC CAA GCA TTC AAT GAG CTC ATC CGA GTG GTC CAC CAG CTG TTG CCG GAA TCC AGC CTC AGG AAG CGG AAA AGG AGT CGC TGC-3′) were generated by subcloning into mRuby2-N1 (54614; Addgene). .. The gRNAs targeting IL-24 or GFP were cloned into a retroviral vector (pMSCV-U6-guide-IRES-Thy1.1) using Gibson’s assembly kit (E2611S; New England Biolabs).

    Transfection:

    Article Title: Bioinformatics Analysis and Experimental Identification of Immune-Related Genes and Immune Cells in the Progression of Retinoblastoma.
    Article Snippet: In vitro and in vivo grade siRNA against SH3GL2 (si-SH3GL2) and nonspecific control siRNA (si-NC) were obtained from RiboBio (Guangzhou, China) and performed according to the manufacturer’s protocol. .. The Y79 cells were transfected with plasmid mRuby2-N1 (empty vector, OE-NC, cat. no. 54614; Addgene, Watertown, MA, USA) or plasmid EndophilinA1-mRuby2 (overexpression SH3GL2, OESH3GL2, cat. no. 171945; Addgene) by Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer’s protocol. ..

    Article Title: Bioinformatics Analysis and Experimental Identification of Immune-Related Genes and Immune Cells in the Progression of Retinoblastoma
    Article Snippet: In vitro and in vivo grade siRNA against SH3GL2 (si-SH3GL2) and nonspecific control siRNA (si-NC) were obtained from RiboBio (Guangzhou, China) and performed according to the manufacturer's protocol. .. The Y79 cells were transfected with plasmid mRuby2-N1 (empty vector, OE-NC, cat. no. 54614; Addgene, Watertown, MA, USA) or plasmid EndophilinA1-mRuby2 (overexpression SH3GL2, OE-SH3GL2, cat. no. 171945; Addgene) by Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer's protocol. ..

    Plasmid Preparation:

    Article Title: Bioinformatics Analysis and Experimental Identification of Immune-Related Genes and Immune Cells in the Progression of Retinoblastoma.
    Article Snippet: In vitro and in vivo grade siRNA against SH3GL2 (si-SH3GL2) and nonspecific control siRNA (si-NC) were obtained from RiboBio (Guangzhou, China) and performed according to the manufacturer’s protocol. .. The Y79 cells were transfected with plasmid mRuby2-N1 (empty vector, OE-NC, cat. no. 54614; Addgene, Watertown, MA, USA) or plasmid EndophilinA1-mRuby2 (overexpression SH3GL2, OESH3GL2, cat. no. 171945; Addgene) by Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer’s protocol. ..

    Article Title: Bioinformatics Analysis and Experimental Identification of Immune-Related Genes and Immune Cells in the Progression of Retinoblastoma
    Article Snippet: In vitro and in vivo grade siRNA against SH3GL2 (si-SH3GL2) and nonspecific control siRNA (si-NC) were obtained from RiboBio (Guangzhou, China) and performed according to the manufacturer's protocol. .. The Y79 cells were transfected with plasmid mRuby2-N1 (empty vector, OE-NC, cat. no. 54614; Addgene, Watertown, MA, USA) or plasmid EndophilinA1-mRuby2 (overexpression SH3GL2, OE-SH3GL2, cat. no. 171945; Addgene) by Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer's protocol. ..

    Article Title: Hijacking of transcriptional condensates by endogenous retroviruses.
    Article Snippet: To knock in the degradation-sensitive FKBPF36V tag at the N-terminus of TRIM28, a repair template containing homology arms spanning upstream and downstream of the target site was cloned into a pUC19 vector (NEB) (Supplementary Fig. 2a). .. The repair template included a mRuby2 fluorescent protein sequence, P2A linker and the FKBP tag sequence (Supplementary Fig. 2a)25. mRuby2 sequence was amplified from the mRuby2-N1 plasmid (Addgene, catalog no. 54614), and the P2A-FKBP sequence was amplified from the PITCh dTAG donor vector (Addgene, catalog no. 91792). .. A guide RNA (Supplementary Table 1b) targeting the N-terminus of TRIM28 was cloned into the sgRNA-Cas9 vector pX458 (Addgene, catalog no. 48138).

    Over Expression:

    Article Title: Bioinformatics Analysis and Experimental Identification of Immune-Related Genes and Immune Cells in the Progression of Retinoblastoma.
    Article Snippet: In vitro and in vivo grade siRNA against SH3GL2 (si-SH3GL2) and nonspecific control siRNA (si-NC) were obtained from RiboBio (Guangzhou, China) and performed according to the manufacturer’s protocol. .. The Y79 cells were transfected with plasmid mRuby2-N1 (empty vector, OE-NC, cat. no. 54614; Addgene, Watertown, MA, USA) or plasmid EndophilinA1-mRuby2 (overexpression SH3GL2, OESH3GL2, cat. no. 171945; Addgene) by Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer’s protocol. ..

    Article Title: Bioinformatics Analysis and Experimental Identification of Immune-Related Genes and Immune Cells in the Progression of Retinoblastoma
    Article Snippet: In vitro and in vivo grade siRNA against SH3GL2 (si-SH3GL2) and nonspecific control siRNA (si-NC) were obtained from RiboBio (Guangzhou, China) and performed according to the manufacturer's protocol. .. The Y79 cells were transfected with plasmid mRuby2-N1 (empty vector, OE-NC, cat. no. 54614; Addgene, Watertown, MA, USA) or plasmid EndophilinA1-mRuby2 (overexpression SH3GL2, OE-SH3GL2, cat. no. 171945; Addgene) by Lipofectamine 3000 transfection reagent (Invitrogen) according to the manufacturer's protocol. ..

    other:

    Article Title: Hijacking of transcriptional condensates by endogenous retroviruses.
    Article Snippet: A guide RNA (Supplementary Table 1b) targeting the N-terminus of TRIM28 was cloned into the sgRNA-Cas9 vector pX458 (Addgene, catalog no. 48138).



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    Comparison of in‐gel fluorescence (IGF) of tagRFP‐T‐, mRuby2‐, mCherry‐, and mKO‐κ‐tagged proteins. (a) Yeast expressing Bmh1‐tagRFP‐T, Bmh1‐mRuby2, Bmh1‐mCherry, or Bmh1‐mKO‐κ were lysed in native conditions, samples were resuspended in LDS sample buffer and incubated for 5 min at the indicated temperatures. After migration on a commercial precast 4%–20%TGX gel (Bio‐Rad), gels were imaged for red fluorescence using a Typhoon or a Chemidoc MP, and total proteins were visualized by the stain‐free technology on a Chemidoc MP. Proteins were then transferred to a nitrocellulose membrane and immunoblotted with anti‐Bmh1 antibodies and then with anti‐rabbit antibodies coupled to HRP, and revealed by chemiluminescence on a Chemidoc MP. * indicates the fluorescent species. (b) Yeast expressing Bmh1‐mCherry were lysed in native conditions, samples were resuspended in LDS sample buffer and incubated for 5 min at the indicated temperatures in a gradient thermocycler. After migration on a commercial precast 4–20% TGX gel (Bio‐Rad), gels were imaged for red fluorescence using a Typhoon, and total proteins were visualized by the stain‐free technology on a Chemidoc. Proteins were then transferred to a nitrocellulose membrane and immunoblotted with anti‐Bmh1 antibodies and then with anti‐rabbit antibodies coupled to HRP, and revealed by chemiluminescence on a Chemidoc MP. (c) Same as (b) on lysates of yeast expressing Bmh1‐mKO‐κ. (d) Quantification of the fluorescence signal as a function of the denaturation temperature for mCherry‐tagged and mKO‐κ‐tagged Bmh1 ( n = 3; ±SD). Solid line: sigmoidal fit of the data (GraphPad Prism), dotted line: 95% confidence interval of the fit. HRP, horseradish peroxidase; LDS, lithium dodecyl sulfate.

    Journal: Biology of the Cell

    Article Title: Direct observation of fluorescent proteins in gels: A rapid, cost‐efficient, and quantitative alternative to immunoblotting

    doi: 10.1111/boc.202400161

    Figure Lengend Snippet: Comparison of in‐gel fluorescence (IGF) of tagRFP‐T‐, mRuby2‐, mCherry‐, and mKO‐κ‐tagged proteins. (a) Yeast expressing Bmh1‐tagRFP‐T, Bmh1‐mRuby2, Bmh1‐mCherry, or Bmh1‐mKO‐κ were lysed in native conditions, samples were resuspended in LDS sample buffer and incubated for 5 min at the indicated temperatures. After migration on a commercial precast 4%–20%TGX gel (Bio‐Rad), gels were imaged for red fluorescence using a Typhoon or a Chemidoc MP, and total proteins were visualized by the stain‐free technology on a Chemidoc MP. Proteins were then transferred to a nitrocellulose membrane and immunoblotted with anti‐Bmh1 antibodies and then with anti‐rabbit antibodies coupled to HRP, and revealed by chemiluminescence on a Chemidoc MP. * indicates the fluorescent species. (b) Yeast expressing Bmh1‐mCherry were lysed in native conditions, samples were resuspended in LDS sample buffer and incubated for 5 min at the indicated temperatures in a gradient thermocycler. After migration on a commercial precast 4–20% TGX gel (Bio‐Rad), gels were imaged for red fluorescence using a Typhoon, and total proteins were visualized by the stain‐free technology on a Chemidoc. Proteins were then transferred to a nitrocellulose membrane and immunoblotted with anti‐Bmh1 antibodies and then with anti‐rabbit antibodies coupled to HRP, and revealed by chemiluminescence on a Chemidoc MP. (c) Same as (b) on lysates of yeast expressing Bmh1‐mKO‐κ. (d) Quantification of the fluorescence signal as a function of the denaturation temperature for mCherry‐tagged and mKO‐κ‐tagged Bmh1 ( n = 3; ±SD). Solid line: sigmoidal fit of the data (GraphPad Prism), dotted line: 95% confidence interval of the fit. HRP, horseradish peroxidase; LDS, lithium dodecyl sulfate.

    Article Snippet: Tagging with yeast codon‐optimized Tag‐RFP‐T or mRuby2 was achieved using the pFa6‐link‐yoTag‐RFP‐T‐CaURA3 and pFA6a‐link‐yomRuby2‐SpHIS5, respectively (gifts from Kurt Thorn and Wendell Lim, Addgene plasmids #44877 and #44843).

    Techniques: Comparison, Fluorescence, Expressing, Incubation, Migration, Staining, Membrane