Construction of IRE1α Mutant Plasmids

pcDNA5/FRT/TO (Invitrogen, Carlsbad, CA) containing IRE1α-HA, wIRE1α (IRE1α-∆434–443 HA) and IRE1α-K907A-HA were described previously (Plumb et al., 2015 (link)). IRE1α-T446A-S450A-T451A-HA mutant was created using previously described primers (Sun et al., 2015 (link)). sIRE1α (IRE1α-S439A-T446A-S450A-T451A-HA), IRE1α-V437A-D443A-HA, IRE1α-∆434-443A-K907A-HA, in pcDNA5/FRT/TO were made by site-directed mutagenesis. Prl-His-2xstrep-IRE1α-FLAG constructs were generated by first inserting Prl-His-2xstrep into pcDNA5/FRT/TO using standard methods. Next, IRE1α-FLAG was amplified beginning from amino acid 29 and cloned into pcDNA5/FRT/TO Prl-His-2xstrep. Mouse spliced XBP1 plasmid (Addgene# 21833) is a kind gift from Dr. David Ron. All PCR reactions were performed with Phusion high fidelity DNA polymerase (NEB, Ipswich, MA), except for site directed mutagenesis, which used Pfu-Ultra polymerase (Agilent technologies, Santa Clara, CA). 3% DMSO was included in all PCR reactions to enhance amplification. The coding regions of all constructs were sequenced to preclude any sequence error. The Yale Keck DNA Sequencing Facility performed all sequencing services.

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Sundaram A., Plumb R., Appathurai S, & Mariappan M. (2017). The Sec61 translocon limits IRE1α signaling during the unfolded protein response. eLife, 6, e27187.

Publication 2017

Phusion high fidelity DNA polymerase Pfu-Ultra polymerase

Amino acid Dmso Dna polymerase Ire1α Mouse Pfu polymerase Plasmid Primers Site directed mutagenesis Xbp1

Corresponding Organization :

Other organizations : Yale University, Universiti Sains Malaysia

Top 5 similar protocols

Variable analysis

independent variables

IRE1α-HA
WIRE1α (IRE1α-Δ434–443 HA)
IRE1α-K907A-HA
IRE1α-T446A-S450A-T451A-HA
SIRE1α (IRE1α-S439A-T446A-S450A-T451A-HA)
IRE1α-V437A-D443A-HA
IRE1α-Δ434-443A-K907A-HA
Prl-His-2xstrep-IRE1α-FLAG

dependent variables

Not explicitly mentioned

control variables

Not explicitly mentioned

positive controls

Mouse spliced XBP1 plasmid (Addgene# 21833)

negative controls

Not explicitly mentioned

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