PCR-amplified human p62 was cloned into pcDNA3.1/hygro(+)-Flag, pcDNA3-HA, or pColdI (His) vector. Human AMPKα1 was cloned into pcDNA3-HA vector. The construction of Flag/V5–rKHK-A or Flag/V5–rKHK-C, GST–KHK-A or GST–KHK-C, Flag-rKHK G257R, His-PRPS1, and KHK short hairpin RNA (shRNA) was described previously (32 (link)). Nrf2 CA (with the deletion of amino acids 1 to 89) was cloned into pcDNA3.1/puro(+)-HA. PINK1 and SQSTM1 shRNA were constructed via ligation of an oligonucleotide targeting human PINK1 (5′-GCTGGAGGAGTATCTGATAGG-3′) and p62 (5′-ACTGGACCCATCTGTCTTCAA-3′) into an Xho I–/Mlu I–digested pGIPZ vector, respectively. Flag-, V5-, or GST-tagged rKHK-A S80A, Flag–rKHK-A S80E, Flag-p62 S28E, Flag-p62 S28A, His–p62 S28A, Flag–p62 K7R, and all the shRNA-resistant p62 constructs containing nonsense mutations of C1017T, T1020C, and G1023A were constructed using the QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Vectors expressing GFP-LC3, HA-ubiquitin-WT, HA-ubiquitin-K48, and HA-ubiquitin-K63 were purchased from Addgene.
Ha ubiquitin wt
Manufactured by Addgene
Sourced in United States
HA-ubiquitin-WT is a protein construct that consists of a Hemagglutinin (HA) tag fused to wild-type ubiquitin. Ubiquitin is a small regulatory protein that can be attached to other proteins, marking them for degradation or altering their function. This construct can be used as a tool in various research applications involving the study of ubiquitination and protein regulation.
Automatically generated - may contain errors
Lab products found in correlation
Ha ubiquitin k63, by Addgene (2 mentions)
Ha ubiquitin k48, by Addgene (2 mentions)
Gfp lc3, by Addgene (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Pcdna3, by Addgene (1 mentions)
Pcmv ha, by Addgene (1 mentions)
Q5 site directed mutagenesis kit, by New England Biolabs (1 mentions)
Pcmv myc, by Addgene (1 mentions)
Flag p53, by Addgene (1 mentions)
Gfp p53, by Addgene (1 mentions)
Flag traf6, by Addgene (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
4 protocols using ha ubiquitin wt
1
Cloning of Protein Constructs
2
Gene Overexpression and Mutation Analysis
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For gene overexpression, the full‐length coding sequences of human CDK1, METTL3, YTHDF2, ACSL4, and UBR5 were chemically synthesized and inserted into pcDNA 3.1 (RRID:Addgene_20 407), pFlag‐CMV‐5a (RRID:Addgene_105 933), pCMV‐Myc (RRID:Addgene_73 365), or pCMV‐HA (RRID:Addgene_15 739) vectors as appropriate. HA‐ubiquitin‐WT was obtained from Addgene (Watertown, MA, USA). Site mutation was conducted using the Q5 Site‐Directed Mutagenesis Kit (#E0554S, New England Biolabs, MA, UK) based on the manufacturer's protocols. All constructs were confirmed by Sanger sequencing. All siRNAs were chemically synthesized by Sangon Biotech (Shanghai, China) and validated. The siRNA targeting sequences are listed in Table S6 (Supporting Information). Cell transfection was carried out using Lipofectamine 3000 reagent (Invitrogen).
3
Investigating p53 and CYLD Interactions
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Flag-P53 was provided by Dr Zhenkun Lou52 (link); HA-CYLD were provided by Dr. Ana Bigas53 (link); CYLD-WT, CYLDR936X and CYLDH871N mutant were provided by Dr. Gilles Courtois8 (link); HA-Ubiquitin-WT (Addgene plasmid 17608), HA-Ubiquitin-K63-only (Addgene plasmid 17606) and HA-Ubiquitin-K48-only (Addgene plasmid 17605) were provided by Dr. Ted Dawson. GFP-p53 (Addgene plasmid 12091) was provided by Dr. T. Jacks. GST-p53 (Addgene plasmid 39479) was provided by Dr. Ie-Ming Shih. PCS2-Flag and pcDNA3-Ha plasmids were used to equalize amount of transfected DNA between samples.
4
Molecular Interactions in Cell Signaling
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The expression vectors for Myc-RIPK1, HA-ubiquitin-WT, HA-ubiquitin-K63, HA-ubiquitin-K48, V5-TRAF2, Flag-TRAF4, Flag-TRAF6 and Flag-BHRF1 were from Addgene (Cambridge, MA, USA). The deletion or mutation constructs of LMP1, RIPK1, and RIPK3 were synthesized by Genechem (Shanghai, China), and all of the plasmids were verified by DNA sequencing. Full-length or truncated cDNAs of LMP1 were cloned into BamHI and EcoRI sites of the GV141 vector (CMV-MCS-3FLAG-SV40-Neomycin). Full-length or truncated cDNAs of RIPK1 and RIPK3 were cloned into KpnI and XhoI sites of the GV219 vector (CMV-MCS-SV40-Neomycin) with an N-terminal Myc tag. The mutRHIM of RIPK1 and RIPK3 were generated by overlap extension PCR to change RIPK1 aa539–542 from IQIG to AAAA and RIPK3 aa458–461 from VQVG to AAAA. Plasmid transfections were performed using Lipofectamine 2000 from Invitrogen according to the manufacturer’s protocol.
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