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 k48
Manufactured by Addgene
Sourced in United States
HA-ubiquitin-K48 is a recombinant protein that contains an HA tag and a ubiquitin molecule with a lysine residue at position 48. It is commonly used as a research tool in cellular and molecular biology studies.
Automatically generated - may contain errors
Lab products found in correlation
Ha ubiquitin k63, by Addgene (2 mentions)
Ha ubiquitin wt, by Addgene (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Gfp lc3, by Addgene (1 mentions)
Pbifc vc155, by Addgene (1 mentions)
Pbifc vn173, by Addgene (1 mentions)
Ha ubiquitin, by Addgene (1 mentions)
Ha ubiquitin k48r, by Addgene (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Flag traf6, by Addgene (1 mentions)
3 protocols using ha ubiquitin k48
1
Cloning of Protein Constructs
2
Plasmid-based Ubiquitin Interaction Analysis
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Plasmid encoding HA‐Ubiquitin, HA‐Ubiquitin‐K48, HA‐Ubiquitin‐K48R were purchased from Addgene and used directly without modification. Plasmid encoding pBiFC‐VC155, pBiFC‐VN173, MDM2‐YFP were purchased from Addgene and used as templates for subcloning. The plasmids were constructed for this study as described in “KEY RESOURCES TABLE” (Supporting Information). All plasmid inserts were validated by sequencing at Genewiz. All siRNAs were purchased from Synbio Technologies. The sequences of the siRNA were described in “KEY RESOURCES TABLE” (Supporting Information). Cultured cells were transfected with different plasmids using Lipofectamine 2000 (Invitrogen, 11668‐019) and with siRNAs using RNAi Max (Invitrogen, 13778150) according to the manufacturer's instructions.
3
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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