FLAG-, Myc-, GFP-, or monomeric red fluorescent protein (mRFP)-tagged PIP5Kα, FLAG- or GFP-tagged PIP5Kα kinase-dead (KD) mutant (D309N, R427Q), and the N-terminal (1–65 aa), catalytic (66–434 aa), CT (435–546 aa), and CT-deleted (1–434 aa; ΔCT) regions of PIP5Kα subcloned into pGEX-6P-1 and/or pcDNA3-FLAG vectors have been described previously [35 (link), 36 (link)]. HA-tagged PIP5Kα, PIP5Kβ, or PIP5Kγ90 expression plasmids were previously provided by Dr. Michael Krauss (Leibniz-Institute for Molecular Pharmacology, Berlin, Germany) [37 (link)]. FLAG- (#11623), HA- (#32836), or GFP-tagged (#84293) Merlin, glutathione S-transferase (GST, #11631)- or FLAG-tagged (#11625) Merlin FERM domain, and FLAG-YAP (#66853), HA-TAZ (#32839), FLAG-TAZ (#27318), HA-NEDD4 (#27002), HA-ubiquitin (#17608), and HA-ubiquitin K48R (#17604) were obtained from Addgene (Cambridge, MA, USA). The F1 (18–98 aa), F2 (111–213 aa), and F3 (221–312 aa) regions of the Merlin FERM domain [23 (link)] were amplified from the GFP-Merlin plasmid by polymerase chain reaction (PCR) and subcloned into the EcoRI–BamHI sites of pEGFP-C2 vector. Myc-Merlin and HA-LATS1 plasmids were provided by Dr. Eunjeong Seo (OliPass Corporation, Yongin, Gyeonggi, Republic of Korea) and HA-Merlin L64P was a gift from Prof. Jung Soon Mo (Ajou University, Suwon, Republic of Korea).
Ha ubiquitin k48r
Manufactured by Addgene
Sourced in United States
HA-ubiquitin K48R is a recombinant protein that contains a hemagglutinin (HA) tag and a lysine (K) to arginine (R) substitution at position 48 of the ubiquitin sequence. This mutation prevents the formation of K48-linked polyubiquitin chains, which are typically associated with protein degradation via the proteasome.
Automatically generated - may contain errors
Lab products found in correlation
Ha ubiquitin, by Addgene (2 mentions)
Ha nedd4, by Addgene (1 mentions)
Ha taz, by Addgene (1 mentions)
Flag taz, by Addgene (1 mentions)
Flag yap, by Addgene (1 mentions)
Pbifc vc155, by Addgene (1 mentions)
Pbifc vn173, by Addgene (1 mentions)
Ha ubiquitin k48, by Addgene (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
2 protocols using ha ubiquitin k48r
1
Comprehensive Toolkit for PIP5Kα Analysis
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.
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