Routine cloning used sequence- and ligation-independent cloning (73). The pcDNA-p62 plasmid (gift from R. Layfield, University of Nottingham) was used for polymerase chain reaction (PCR) (Phusion polymerase, New England Biolabs) to generate the retroviral pMXsIP-p62 plasmid. The cytomegalovirus (CMV) promoter from pEYFP-C1 (Clontech), 3×Flag at N terminus (from p3Tag1 vector, Agilent), and hemagglutinin (HA)–tag at C terminus (YPYDVPDYA introduced by PCR) were added to generate the pMXCMV-flag-p62-HA plasmid. However, as we and others have noted the cleavage of HA-tag by caspases (53, 74), anti-HA Western blots were not used in this study to detect Flagp62HA. The pIRES-Puro2 caspase-8 plasmid was a gift from R. Eils, German Cancer Research Centre (75). pGEX-4T1-p62 (gift from R. Layfield) was used to generate pGEX-6P1-p62297–440. Site-directed mutagenesis used single oligonucleotide-based linear PCR to generate Asp329 (76). Various p62 truncation variants were generated by PCR (regions shown in fig. S1A). The LIR mutant was p62D337A/W338A/L341A, and the KIR mutant was p62T350A. The Myc-RIPK1 was a gift from X. Lin (Addgene plasmid #44159). pMXCMV-YFP-stop, where YFP expression is driven by a CMV promoter (from pEYFP-C1, Clontech), was used as a control plasmid when carrying out retroviral transductions.

A TEV protease site was introduced into the pMXCMV-Flag-p62-HA plasmid by PCR to replace the caspase-8 site: 326MESD↓N330 was replaced by MENLYFQ↓GN (TEV recognition sequence in italics; ↓ indicates protease-cleavage site). An all-in-one lentiviral plasmid pLTREK3-TEV-T2A-GFP for doxycycline-inducible TEV-T2A-GFP and constitutive rtTA3 expression was generated as follows. The TRE-XTight promoter (from pRetroX-Tight-Puro; Clontech) was cloned into the pLentiV2 vector upstream of a TEV-T2A-GFP cassette; the self-cleaving T2A linker peptide generates separate TEV protease and GFP proteins. PGK promoter (from pRetroX-Tight-Puro) and rtTA3 (from pTripZ; Dharmacon) were cloned downstream of TEV-T2A-GFP. Stable pMXCMV-flag-p62TEV-HA transduction was achieved using puromycin selection, and flow sorting of GFP+ cells provided Flagp62TEV- and TEV-T2A-GFP–expressing Δp62 HeLa cells (Δp62/Flagp62TEV+TEV-T2A-GFP cells). All DNA fragments cloned by PCR were validated by sequencing (GATC Biotech).

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.