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. 2022 Jun 28;41(15):e109566. doi: 10.15252/embj.2021109566
Reagent/Resource Source Identifier
Chemicals, peptides, and Recombinant proteins
UBE1 Boston Biochem Cat. # E‐304
UBE2D1 Boston Biochem Cat. # E2‐616
GST UbcH5a/UBE2D1 Boston Biochem Cat. # E2‐615
UBE2D2 Boston Biochem Cat. # E2‐622
UBE2D3 Boston Biochem Cat. # E2‐627
UBE2N/Uev1a Boston Biochem Cat. # E2‐664
UBE2W Boston Biochem Cat. # E2‐740
10X E3 Ligase Reaction Buffer Boston Biochem Cat. # B‐71
10X Ubiquitin conjugation Reaction Buffer Boston Biochem Cat. # B‐70
10X Energy Regeneration Solution Boston Biochem Cat. # B‐10
Ubiquitin Boston Biochem Cat. # U‐100H
FLAG (DYKDDDDK)‐Ubiquitin Boston Biochem Cat. # U‐120
UbNoK Boston Biochem Cat. # UM‐NOK
Ub3KTR Boston Biochem Cat. # UM‐3KTR
M1linked‐ linear ubiquitin UbiQ Cat. # UbiQ‐L01
UBE2D1 ubiquitin charged Boston Biochem Cat. # E2‐800
N‐Ethylmaleimide (NEM) Sigma‐Aldrich Cat. # E3876
cOmplete™, EDTA‐free Protease Inhibitor Cocktail Roche Cat. # 11873580001
MG132 Selleckchem Cat. # S2619
Apyrase Sigma‐Aldrich Cat. # A2230
Alkaline Phosphatase Yellow (pNPP) Liquid Substrate Sigma‐Aldrich Cat. # P7998
Q5 Site‐Directed Mutagenesis Kit NEB Cat. # E0552S
4x Laemmli Sample Buffer Bio‐Rad Cat. # 1610747
β‐Mercaptoethanol Sigma‐Aldrich Cat. # M6250
Dynabeads™ Co‐Immunoprecipitation Kit Invitrogen Cat. # 14321D
Pierce™ Anti‐DYKDDDDK Magnetic Agarose Invitrogen Cat. # A36797
RediStain™ WormDye Lipid Green NemaMetrix Cat. # DYE9439
Rapid Gold BCA Protein Assay Kit Thermo Fisher Scientific Cat. # A53225
IMPACT™ Kit NEB Cat. # E6901S
AHCY‐1::6xHis This paper
CHN‐1 This paper
6xHis::SUMO::CHN‐1 This paper
CHN‐1Δ110 This paper
CHN‐1Δ87 This paper
CHN‐1Δ95 This paper
CHN‐1R230A This paper
UFD‐2 This paper
UFD‐2EEVD This paper
6xHis::UFD‐2 This paper
6xHis::UFD‐2P951A Ackermann et al (2016)
6xHis::UFD‐2ΔUbox This paper
6xHis::Ufd2p This paper
HSP‐1::6xHis This paper
6xHis::DAF‐21 This paper
HSP‐1ΔEEVD::6xHis This paper
6xHis::DAF‐21ΔEEVD This paper
HSP‐1EEYD::6xHis This paper
Antibodies
Anti‐CHN‐1 antibody Tawo et al (2017)
Anti‐UFD‐2 antibody Ackermann et al (2016)
Anti‐AHCY‐1 antibody This study
Anti‐Ubiquitin antibody Cell signaling Technology Cat. # 3936s
Anti‐Histidine antibody Santa Cruz Biotechnology, Inc. Cat. # SC‐53073
Anti‐GST antibody Sigma‐Aldrich Cat. # G1160
Anti‐UBE2D1 antibody Biorad Cat. # VPA00296
Bacterial Strains
E. coli RNAi feeding strain Caenorhabditis Genetics Center HT115(DE3)
E. coli feeding strain Caenorhabditis Genetics Center OP50
Ahringer RNAi library Source BioScience C. elegans RNAi Collection (Ahringer)
Rosetta™ 2 (DE3) Novagen Cat. # 71400
BL21 Star™ (DE3) Thermo Fisher Scientific Cat. # C601003
Top10 Thermo Fisher Scientific Cat. # C4040
Oligonucleotides

pTYB21‐MBP::Intein‐UFD‐2

Forward: GGTGGTTGCTCTTCCAACATGATTGAAGACGAGAAAGCAGG

Reverse: GGTGGTCTGCAGTCATTATTTCTTTGAATTTCTTT

 This paper

pET‐6xHis::SUMO‐CHN‐1Δ110

Forward: ATTGAGAACGCCCTCAAAC

Reverse: GCTAGCTAGACCACCAATC

 This paper

pET‐6xHis::SUMO‐CHN‐1Δ87

Forward: TACAGTGAAGCAATAAGCTG

Reverse: GCTAGCTAGACCACCAATC

 This paper

pET‐6xHis::SUMO‐CHN‐1Δ95

Forward: TCCAAAGCGCTCTACCAT

Reverse: GCTAGCTAGACCACCAATC

 This paper

pET‐21a‐VSV‐HSP‐1ΔEEVD::6xHis

Forward: GCGGCCGCACTCGAG

Reverse: TCCTCCGGCGGCTCCTCC

 This paper

pET‐21a‐6xHis::DAF‐21ΔEEVD

Forward: TAATGAGGATCCGAATTCGAG

Reverse: CTCAGCTCCCTCAATCTT

 This paper

pET‐6xHis::SUMO‐CHN‐1R230A

Forward: TCCAGTCACAGCAAAACCACTTAC

Reverse: TCGAAATGGCCAATTCTTC

 This paper

pTYB21‐MBP::Intein‐UFD‐2 Y907V(EEVD)

Forward: GAAGAAGAGGTTGATGATGTACCA

Reverse: TGGTACATCATCAACCTCTTCTTC

 This paper

pET‐21a‐VSV‐HSP‐1 V639Y(EEYD)

Forward: ATCGAGGAGTACGACGCGGCC

Reverse: GGCCGCGTCGTACTCCTCGAT

 This paper
Recombiant DNA
pET28a‐6xHis::Ufd2p Liu et al (2017)
pET‐6xHis::SUMO::CHN‐1 This paper
pET28a‐6xHis::UFD‐2 This paper
pLATE31‐AHCY‐1::6xHis This paper
pET21a‐VSV::HSP‐1::6xHis This paper
pET21a‐6xHis::DAF‐21 This paper
pTYB21‐MBP::Intein‐UFD‐2 This paper
pET‐6xHis::SUMO‐CHN‐1Δ110 This paper
pET‐6xHis::SUMO‐CHN‐1Δ87 This paper
pET‐6xHis::SUMO‐CHN‐1Δ95 This paper
pET‐21a‐VSV‐HSP‐1EEYD::6xHis This paper
pET‐21a‐VSV‐HSP‐1ΔEEVD::6xHis This paper
pET‐21a‐6xHis::DAF‐21ΔEEVD This paper
pET‐6xHis::SUMO‐CHN‐1R230A This paper
pTYB21‐MBP::Intein‐UFD‐2 Y907V(EEVD) This paper
pET‐21a‐VSV‐HSP‐1 V639Y(EEYD) This paper
Experimental models: Organisms/Strains
C. elegans: Bristol (N2) strain as wild‐type CGC N/A
C. elegans: chn‐1(by155)I CGC WormBase ID: WBVar00000641
C. elegans: unc‐119(ed4)III; hhIs136[unc‐119(+); chn‐1p::chn‐1::FLAG] Tawo et al (2017) N/A
C. elegans: ufd‐2(tm1380)II CGC WormBase ID: WBVar00250374
C. elegans: chn‐1(by155)I; ufd‐2(tm1380) II This paper N/A
C. elegans: chn‐1(by155)I; ahcy‐1(syb646[ahcy‐1::GFP]I This paper N/A
C. elegans: ahcy‐1(syb646[ahcy‐1::GFP]I; ufd‐2(tm1380)II; This paper N/A
C. elegans: unc‐119(ed4)III; hhIs136[unc‐119(+); chn‐1p::chn‐1::FLAG]; ahcy‐1(syb646[ahcy‐1::GFP]I This paper N/A
Software and Algorithms
Graph Pad Prism Graph Pad Software, Inc. www.graphpad.com
Image Lab™ Version 6.0.0 build 25 Bio‐Rad Laboratories, Inc. www.bio‐rad.com/de‐de/product/image‐lab‐software?ID=KRE6P5E8Z
ImageJ 1.53c Wayne Rasband, NIH, USA www.imagej.nih.gov/ij
Materials
Nunc MaxiSorp™ flat‐bottom Thermo Fisher Scientific Cat. # 44‐2404
Hiload 16/600 Superdex S200 GE Healthcare Cat. # GE28‐9893‐35