FLAG-tagged LGP2, RIG-I, MDA5, GFP, His-tagged LGP2, HA-tagged LGP2 plasmids are described elsewhere [8 (link)]. FLAG-tagged Dicer was generous gifts from Dr. V. N. Kim (Seoul National University). mIFNβ-luc plasmid was generous gift from Dr. J. Marques (Universidade Federal de Minas Gerais). IMAGE clone of TRBP (IMAGE 3162979; GenBank: BC002419.2 ) is purchased from Thermo Scientific. The open reading frame of TRBP was PCR amplified with restriction enzyme sites and ligated to multiple cloning sites of pB42AD (Clontech), pLexA (Clontech), p3 × FLAG CMV10 (Sigma-Aldrich), pcDNA3 HA (Thermo Fisher Scientific, Waltham, MA) vectors. The coding region of LGP2 is PCR amplified with restriction enzyme sites and ligated to pLexA (Clontech) and pB42AD (Clontech).
Plexa
Manufactured by Takara Bio
The PLexA is a compact and automated cell culture system designed for high-throughput screening and analysis. It features precise control over culture parameters, automated liquid handling, and integrated detection capabilities to support a wide range of cell-based assays.
Automatically generated - may contain errors
4 protocols using plexa
1
Plasmid Generation and Cloning
2
Yeast Two-Hybrid Protein Interaction Assay
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The full-length sequences of Bam, Brat and, AGO1 full length were cloned into various sites of pLex A and pACT2 (Clontech). These combinations of plasmids expressing the Lex A-fused protein and GAD-fused protein were cotransformed into the yeast strain YPH500 (MAT ade 2, his3, leu 2, lys2, trp1, ura3) harboring the pSH18-34 plasmid (lexAop-LacZ reporter) by the standard lithium acetate method (Ito et al., 1983; Gyuris et al., 1993). The independent transformants were patched onto glucose plates containing X-gal for 24–48 h and followed by liquid assay and reported as beta-galactosidase units.
3
Molecular cloning of Brat and Bam
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The S2 cell expression vectors, pAc5.1A-FLAG- Bam and pAc5.1A-Myc-Bgcn were
obtained from the previous studies (Kim et al.,
2010 ). The NHL domain (aa 724- 1037), the N-terminus (aa 1-730), and
the full-length of Brat (LD28374 from the Drosophila Genomics
Resource Center) were cloned into the NotI/XbaI site of pAc5.1A-Myc to generate
pAC5.1A-Myc-Brat NHL and a pAC5. 1A-Myc-Brat N terminal, and the
pAC5.1A-Myc-Brat FL tagged proteins in S2 cells. For yeast two-hybrid, the full-
length of Bam was cloned into Bam HI/XhoI sites of pLexA (Clontech) to generate
LexA-Bam. The N- terminus and full length of Brat were cloned into the Bam HI/
SacI and Bam HI/XhoI, respectively, of pACT2 (Clontech) to generate TAD-Brat FL
and -Brat N. The Brat NHL domain (aa 724-aa 1037) was cloned into SalI/Xba1 of
pGAD 424 (Clontech) to generate TAD-Brat NHL. For the fragment complementation
assay in HEK293T cells, the coding sequence and its derivatives of Bam and Brat
were cloned into Bam HI/XhoI and the KpnI/HindIII site of the vectors
(CoralHue_Fluo-chase kit; Medical and Biological Lab Co.), respectively, to
produce protein fused to either the N- or the C- terminal fragment of the
monomeric Kusabira-Green (mKG). P50- mKG_N and P65-mKG_C were provided as the
control.
obtained from the previous studies (
2010
the full-length of Brat (LD28374 from the Drosophila Genomics
Resource Center) were cloned into the NotI/XbaI site of pAc5.1A-Myc to generate
pAC5.1A-Myc-Brat NHL and a pAC5. 1A-Myc-Brat N terminal, and the
pAC5.1A-Myc-Brat FL tagged proteins in S2 cells. For yeast two-hybrid, the full-
length of Bam was cloned into Bam HI/XhoI sites of pLexA (Clontech) to generate
LexA-Bam. The N- terminus and full length of Brat were cloned into the Bam HI/
SacI and Bam HI/XhoI, respectively, of pACT2 (Clontech) to generate TAD-Brat FL
and -Brat N. The Brat NHL domain (aa 724-aa 1037) was cloned into SalI/Xba1 of
pGAD 424 (Clontech) to generate TAD-Brat NHL. For the fragment complementation
assay in HEK293T cells, the coding sequence and its derivatives of Bam and Brat
were cloned into Bam HI/XhoI and the KpnI/HindIII site of the vectors
(CoralHue_Fluo-chase kit; Medical and Biological Lab Co.), respectively, to
produce protein fused to either the N- or the C- terminal fragment of the
monomeric Kusabira-Green (mKG). P50- mKG_N and P65-mKG_C were provided as the
control.
4
Yeast Two-Hybrid Screening of BnaTIRs/AFBs and BnaIAA7/ARF
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For BnaTIRs/AFBs interaction assays, ED1, ed1, BnaA03.IAA7, and BnaA05.IAA7 full-length cDNAs were cloned into pB424AD (Clontech), while BnaTIRs/AFBs full-length cDNA were cloned into pLexA (Clontech). BnaIAA7/TIRs or AFBs were transformed into the EGY48A strain and selected on control media plates (SD/−Ura/−His/−Trp) for 3 days at 30 °C. Three single clones were then selected by spotting on a selective plate (SD/Gal/Raff/−Ura–His–Trp + X–Gal + BU salts) by incubation for 3 days. For BnaIAA7 and BnaARF interaction assays, the three BnaIAA7 full-length cDNAs were cloned into pGBKT7 (Clontech), and copies of BnaARF full-length cDNA were cloned into pGADT7 (Clontech). The BnaIAA7/ARF plasmids were then transformed into AH109 strain and selected on control media (SD/–Leu–Trp) plate by incubation for 3 days at 30 °C. The interactions were detected on the selective plate (SD/−Leu−Trp−His−Ade + X-a−Gal). Primers for PCR amplification are listed in Supplementary Table 6.
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