Journal: Bioengineered

Article Title: Circular RNA-UBE2D2 accelerates the proliferation and metastasis of non-small cell lung cancer cells via modulating microRNA-376a-3p/Eukaryotic Translation Initiation Factor 4γ2 axis.

doi: 10.1080/21655979.2022.2027068

Figure Lengend Snippet: Figure 5. Circ-UBE2D2 elevates EIF4G2 via miR-376a-3p. A: Binding sites of miR-376a-3p and circ-UBE2D2; B/C: Test of miR-376a-3p in A549 cells of each group was via RT-qPCR; D: Verification of the targeting of circ-UBE2D2 with miR-376a-3p was via the dual luciferase digestion report experiment; E: Examination of EIF4G2 in LC tissue and para-cancerous tissues was via RT-qPCR (N = 48); F: The correlation was of circ-UBE2D2 with EIF4G2; G: The association was of miR-376a-3p with EIF4G2. The data in the figure were all measurement data, and representation of the values was as mean ± SD.

Article Snippet: The primary antibody EIF4G2 (1:2000, 2182, Cell Signaling Technology), GAPDH (1:1000, 2118) and secondary horseradish peroxidase conjugated antibody (1:2000, 7074, Cell Signaling Technology) were adopted.

Techniques: Binding Assay, Quantitative RT-PCR, Luciferase

Journal: Bioengineered

Article Title: Circular RNA-UBE2D2 accelerates the proliferation and metastasis of non-small cell lung cancer cells via modulating microRNA-376a-3p/Eukaryotic Translation Initiation Factor 4γ2 axis.

doi: 10.1080/21655979.2022.2027068

Figure Lengend Snippet: Figure 6. Circ-UBE2D2 facilitates the progression of NSCLC via modulating miR-376a-3p/EIF4G2 axis. A: Test of EIF4G2 in A549 cells of each group was via RT-qPCR and Western blot; B-C: Examination of the cell proliferation was via CCK-8 and plate cloning; D: Test of the cell apoptosis was via flow cytometry; E: Detection of the cell migration was via plate scratch; F: Examination of the cell invasion was via Transwell. The data in the figure were all measurement data, and representation of the values was as mean ± SD, *P < 0.05 versus the si-UBE2D2 + pcDNA-NC.

Article Snippet: The primary antibody EIF4G2 (1:2000, 2182, Cell Signaling Technology), GAPDH (1:1000, 2118) and secondary horseradish peroxidase conjugated antibody (1:2000, 7074, Cell Signaling Technology) were adopted.

Techniques: Quantitative RT-PCR, Western Blot, CCK-8 Assay, Cloning, Flow Cytometry, Migration

Journal: Molecular cell

Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

doi: 10.1016/j.molcel.2018.09.020

Figure Lengend Snippet: Figure 2. SDS22-PP1-I3 Needs to Be Disassembled by p97 to Promote Association of PP1 with Substrate-Specifying Subunits and Full Phos- phatase Activity (A) Increased binding of a p97 substrate-trapping mutant to PP1, SDS22, and I3. p97 wild-type (WT) or the E578Q mutant were expressed in stable cell lines and isolated. Indicated proteins were detected by western blot. (B–D) Chemical inhibition of p97 by NMS-873 (10 mM) blocks SDS22-PP1 dissociation (B), I3-PP1 dissociation (C), and NIPP1-PP1 association (D). For (D), NMS- 873 was added already during the pulse. Immunoprecipitations as indicated, detection of radiolabeled PP1, and quantification are shown. Shown are means ± SD; n = 3. (E) Blocking SDS22 dissociation from PP1 and NIPP1 and MYPT1 association to PP1 by p97 inhibition is reversible. Cells were treated with NMS-873 (NMS, 10 mM) during pulse radiolabeling and then chased for 1 hr with either DMSO or NMS, followed by another washout of NMS for 1 hr. IPs as indicated and autoradiography of PP1 are shown. (F) Inhibition of p97 during PP1 biogenesis affects PP1 activity. Strep-tagged PP1g was shortly induced in stable cell lines. Cells were chased in cycloheximide plus NMS-873 (5 mM) or vehicle alone. Tagged PP1g was isolated and trypsin-revealed phosphatase activity determined (DPM, decays per minute). Strep-tagged GFP served as control. Dotted line indicates fraction of activity attributed to background expression of PP1g-Strep (see also G). Note the increase of activity during the chase, which is abrogated by NMS-873. Shown are means ± SEM. n = 5; *p < 0.05; **p < 0.01 as determined by repeated-measures ANOVA and Bonferroni’s multiple comparisons post hoc test. (G) Western blot of samples analyzed in (F). Note background expression of PP1g-strep in uninduced cells. See also Figure S2.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

Techniques: Activity Assay, Binding Assay, Mutagenesis, Stable Transfection, Isolation, Western Blot, Inhibition, Blocking Assay, Radioactivity, Autoradiography, Control, Expressing

Journal: Molecular cell

Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

doi: 10.1016/j.molcel.2018.09.020

Figure Lengend Snippet: Figure 3. The SEP Domain Adapters p37, p47, and UBXN2A Assist p97 in SDS22-PP1-I3 Disassembly (A) Domain structure of human p97 adapters that share a SEP domain of unknown function. The UBX domain and the SHP box mediate interaction with p97. Only p47 contains a ubiquitin-binding UBA domain. (B) Strep-Tactin pull-downs of indicated strep-hemagglutinin (HA)-tagged (SH) SEP domain adapters and western blot with indicated antibodies. Asterisk indicates an unspecific band detected by the SDS22 antibody. (C) p37, p47, and UBXN2A function partially redundantly as p97 adapters for SDS22-PP1-I3. p47 knockout (KO) or parental cells were treated with indicated siRNAs. p97 was immunoprecipitated and indicated associated proteins detected by western blot. Npl4 was probed as alternative p97 adapter control. (D) Partially redundant roles in PP1 complex disassembly. Autoradiography of pulse-chase experiments in p47 KO or parental HeLa cells combined with siRNA- mediated knockdown of p37 and UBXN2A or control depletion as indicated. (E) Quantification of (D). Shown are means ± SD; n = 3. (F) Loss of SEP domain adapters causes a shift in the PP1 interaction landscape. PP1 was isolated from p47 KO cells after depletion of p37 and UBXN2A or from control-depleted parental cells. Associated proteins were analyzed by quantitative mass spectrometry and results compared in a volcano plot. The black line indicates the threshold for significant differences between treatment conditions (false discovery rate [FDR] < 0.05; s0 = 0.1). Established direct PP1-interacting proteins (Heroes et al., 2013) are marked in black circles, of which those discussed in the text are labeled (closed circles). (G) Indicated proteins from (F) were validated by western blot. (H) Requirement of SEP domain adapters for cell viability and proliferation. Cell populations were treated as indicated and subjected to the 3-(4,5-dimethylthiazol- 2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay at indicated time points. Shown are means ± SD of one represen- tative experiment with technical triplicates. (I) Loss of adapters induces apoptosis. Lysates of indicated cell populations were subjected to western blot analysis to monitor poly ADP-ribose poly- merase 1 (PARP-1) and caspase-3 cleavage. See also Figure S3 and Table S1.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

Techniques: Ubiquitin Proteomics, Binding Assay, Western Blot, Knock-Out, Immunoprecipitation, Control, Autoradiography, Pulse Chase, Knockdown, Isolation, Mass Spectrometry, Labeling, MTS Assay

Journal: Molecular cell

Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

doi: 10.1016/j.molcel.2018.09.020

Figure Lengend Snippet: Figure 4. The p37 Adapter Recruits p97 to the SDS22-PP1-I3 Complex by Direct Binding of the SEP Domain to I3 (A) Cartoon structure of p37 mutant proteins used here. The asterisk indicates SHP box mutations that interfere with p97 binding. (B) Direct binding of p97-p37 to SDS22-PP1-I3 requires the p37 SEP domain and interaction between p97 and p37. The SDS22-PP1-I3 complex generated in insect cells was incubated with p97 and p37 or indicated p37 mutants. PP1 was isolated and associated proteins analyzed by western blot. (C) Homology modeling of p37 based on the p47 SEP domain structure (PDB: 1SS6). Positions of genetically encoded crosslink amino acids are indicated. (D) I3, but not SDS22 or PP1, forms crosslinks with residue 182 in the SEP domain of p37. SDS22-PP1-I3 was incubated with p97 and the p37-L182pBPA variant, UV irradiated as indicated, and processed for western blotting with indicated antibodies. (E) Experiments as in (D) with p37 crosslink variants L182pBPA or F89pBPA and indicated components. (F) p97-p37 binding to the PP1 complex depends on I3. SDS22-PP1 and I3 were generated separately. Binding assays with SDS22-PP1 in the presence or absence of I3 or I3mut with mutations in the RVXF motif that abrogate PP1 binding are shown. (G) Model for recruitment of p97 to the PP1 complex. S, SEP domain; U, UBX domain. See also Figure S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

Techniques: Binding Assay, Mutagenesis, Generated, Incubation, Isolation, Western Blot, Residue, Variant Assay, Irradiation

Journal: Molecular cell

Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

doi: 10.1016/j.molcel.2018.09.020

Figure Lengend Snippet: Figure 5. Reconstitution of SDS22-PP1-I3 Disassembly by p97-p37 with Pure Components in the Absence of Ubiquitination (A) Rapid PP1 subunit exchange at sub-stoichiometric concentrations of p97. Purified SDS22-PP1-I3 was incubated with NIPP1 and p97-p37 at the indicated molar ratios in the presence of ATP or ATPgS. Disassembly and exchange to NIPP1 was followed over time by co-immunoprecipitation of PP1g. (B) Reactions were carried out as in (A) in the presence or absence of ATP, ATPgS, or p37 as indicated and separated by size-exclusion chromatography. Note co-migration of the PP1 complex with the p97 hexamer in the presence of ATPgS dependent on p37 and disassembly of the PP1 complex to monomers with ATP. (C) p37 function depends on the SEP domain. Disassembly reactions as in (A) were carried out with p97 (3 nM) and p37 wild-type (wt) or p37 DSEP (50 nM). (D) I3 binding to PP1 is required for SDS22-PP1 disassembly. Reactions in the presence or absence of I3 or the PP1 binding-deficient I3mut are shown. See also Figure S5.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

Techniques: Ubiquitin Proteomics, Incubation, Immunoprecipitation, Size-exclusion Chromatography, Migration, Binding Assay

Journal: Molecular cell

Article Title: Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

doi: 10.1016/j.molcel.2018.09.020

Figure Lengend Snippet: Figure 6. PP1 Complex Disassembly Involves ATPase-Driven Pulling of I3 into the Central Channel of p97 and Concomitant Unfolding (A) Positions of genetically encoded crosslink amino acids at the pore loops of D1 (E314pBPA) or D2 (D592pBPA) within the channel of the p97 hexamer. (B) p97 variants harboring the indicated crosslink amino acids were UV activated during disassembly reactions and crosslink products analyzed by western blot with indicated antibodies (WB). Note that the signal at the top of the gel likely corresponds to multiple copies of p97 crosslinked to I3 and to each other. (C) Crosslinks were carried out in the presence of ATP or ATPgS with or without p37 as indicated. Note that I3 crosslinks to D1 and D2 depended on p37 and that D2 crosslinks were suppressed by ATPgS. (D) Unfolding of a reporter domain on I3. A complex of SDS22, PP1, and I3 fused to Eos was incubated with different concentrations of p97, p37, or Ufd1-Npl4 and ATP or ATPgS as indicated. Eos fluorescence was monitored by spectrometry. A peptide backbone break in Eos was induced beforehand to prevent refolding. (E) Unfolding depends on binding of p37 to I3 and to p97. Experiments as in (D) with indicated p37 variants are shown. See also Figure S6.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER sip47 s1: AGCCAGCUCUUCCAUCUUATT Microsynth N/A sip37 s1: GUGCCGUAAUAUAGAGGAATT Microsynth N/A sip37 s2: CAGUUUAGAUGAUGGAGAATT Microsynth N/A siUBXN2A s1: AGAAGAGGUGGACGUUAAATT Microsynth N/A siUBXN2A s2: GAAAUAUGUUUGUCUACGATT Microsynth N/A siPP1 Santa Cruz sc-43545 Recombinant DNA p47 CRISPR/Cas9 KO plasmid Santa Cruz sc-402328 p47 HDR plasmid Santa Cruz sc-402328-HDR pEVOL-pBpF plasmid Addgene #31190 pcDNA5FRT/TO-p37-Strep-HA H€ulsmann et al., 2018; Addgene #113485 pcDNA5FRT/TO-p47-Strep-HA H€ulsmann et al., 2018; Addgene #113475 pcDNA5FRT/TO-UBXN2A-Strep-HA H€ulsmann et al., 2018; Addgene #113480 pcDNA5FRT/TO-UBXN11-Strep-HA H€ulsmann et al., 2018; Addgene #113493 pcDNA5FRT/TO-Ufd1-Strep-HA H€ulsmann et al., 2018; Addgene #113474 pcDNA5/FRT/TO/GFP-SH R. Aebersold; H€ulsmann et al., 2018 N/A pGEX-6P-1 p37 This study; Addgene #113500 pGEX-6P-1 p37deltaSEP This study; Addgene #113501 pGEX-6P-1 p37deltaN This study; Addgene #113502 pGEX-6P-1 p37 SHPmut This study; Addgene #113503 pGEX-6P-1 p37 deltaUBX This study; Addgene #113504 pGEX-4T-1 UBXN2A This study; Addgene #113505 pcDNA5FRT/TO PP1gamma-2*Strep This study; Addgene #113506 pET15b His-p97 This study; Addgene #113507 pET15b p97-Strep-His E314Amb This study; Addgene #113508 pET15b p97-His D592Amb This study; Addgene #113509 pFL His-p97 This study; Addgene #113510 pFL His-SDS22 / PP1gamma This study; Addgene #113511 pFL His-I3 This study; Addgene #113513 pFL I3 This study; Addgene #113514 pFL His-I3 V41A / W43A This study; Addgene #113515 pFL His-mEos3.2-I3 This study; Addgene #113516 Software and Algorithms FlowJo v10.5.0 FlowJo, LLC https://www.flowjo.com/ OriginPro 2016G OriginLab https://www.originlab.com/ MaxQuant v1.5.3.30 and the MaxLFQ algorithm Cox et al., 2014 http://www.biochem.mpg.de/ 5111795/maxquant Perseus v1.5.5.3 Tyanova et al., 2016 http://www.biochem.mpg.de/ 5111810/perseus SigmaPlot v12.5 Systat Software http://www.systat.de/ SigmaPlot_Produktseite.html PyMol Schrödinger, LLC https://www.pymol.org/2/ Cell Profiler Kamentsky et al., 2011 http://www.cellprofiler.org/

Techniques: Western Blot, Incubation, Binding Assay

Journal: Cell Reports

Article Title: The protease SPRTN and SUMOylation coordinate DNA-protein crosslink repair to prevent genome instability

doi: 10.1016/j.celrep.2021.110080

Figure Lengend Snippet:

Article Snippet: Rabbit anti-p97 , Proteintech , Cat#10736-1-AP; RRID: AB_2214635.

Techniques: Virus, Subcloning, Recombinant, Staining, Picogreen Assay, Proliferation Assay, Flow Cytometry, DNA Extraction, Sequencing, Luciferase, Software, Transfection, Modification, Magnetic Beads, Membrane

Journal: Journal of Biological Chemistry

Article Title: The UBX Protein SAKS1 Negatively Regulates Endoplasmic Reticulum-associated Degradation and p97-dependent Degradation

doi: 10.1074/jbc.m110.158030

Figure Lengend Snippet: FIGURE 1. SAKS1 interacts with hexameric p97. A, schematic of SAKS1 domain structure and mutants. B, immunoprecipitations (IP) were performed from HEK-293A cells with antibodies versus SAKS1, p97, or a control antibody. The lysate represents 5% input. Endogenous SAKS1 and p97 co-immunoprecipi- tate. C, SAKS1 and His6-p97 were purified from bacteria and analyzed by gel filtration alone and together. SAKS1 co-migrates with hexameric p97.

Article Snippet: All point-mutated constructs were generated using the QuikChange site-directed mutagenesis kit (Stratagene). pQE9-His-p97 (wild type), ubiquitin-G76V-GFP, and pGEX hHR23A were obtained from Addgene (Addgene plasmids 14666, 11941, and 10864) (23–25).

Techniques: Control, Purification, Bacteria, Filtration

Journal: Journal of Biological Chemistry

Article Title: The UBX Protein SAKS1 Negatively Regulates Endoplasmic Reticulum-associated Degradation and p97-dependent Degradation

doi: 10.1074/jbc.m110.158030

Figure Lengend Snippet: FIGURE 3. Generation of a SAKS1 mutant deficient in p97 binding. A, indicated resin-bound GST constructs were tested for binding to recom- binant p97 in vitro. The resulting complexes were run on SDS-PAGE and stained with the protein stain IRDye. B, HEK-293A cells transfected with the indicated constructs were subjected to FLAG immunoprecipitations (IP) and blotted for the FLAG construct as well as endogenous p97. The amount of p97 precipitated relative to the wild-type construct (middle lane) is shown.

Article Snippet: All point-mutated constructs were generated using the QuikChange site-directed mutagenesis kit (Stratagene). pQE9-His-p97 (wild type), ubiquitin-G76V-GFP, and pGEX hHR23A were obtained from Addgene (Addgene plasmids 14666, 11941, and 10864) (23–25).

Techniques: Mutagenesis, Binding Assay, Construct, In Vitro, SDS Page, Staining, Transfection

Journal: Journal of Biological Chemistry

Article Title: The UBX Protein SAKS1 Negatively Regulates Endoplasmic Reticulum-associated Degradation and p97-dependent Degradation

doi: 10.1074/jbc.m110.158030

Figure Lengend Snippet: FIGURE 5. SAKS1 p97 and polyubiquitin binding mutants do not impact ERAD. A, HEK-293A cells were co-transfected with NHK-HA and either FLAG- SAKS1 constructs or control DNA. Lysates from a cycloheximide (CHX) chase were analyzed by Western blotting. B, results of several replicates of experi- ments performed as in A were quantified and graphed. The mutants do not significantly differ from the control at any time point, p 0.01.

Article Snippet: All point-mutated constructs were generated using the QuikChange site-directed mutagenesis kit (Stratagene). pQE9-His-p97 (wild type), ubiquitin-G76V-GFP, and pGEX hHR23A were obtained from Addgene (Addgene plasmids 14666, 11941, and 10864) (23–25).

Techniques: Binding Assay, Transfection, Construct, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The UBX Protein SAKS1 Negatively Regulates Endoplasmic Reticulum-associated Degradation and p97-dependent Degradation

doi: 10.1074/jbc.m110.158030

Figure Lengend Snippet: FIGURE 6. Polyubiquitin increases the association between SAKS1 and p97. A, HEK-293A cells transfected with the indicated constructs were subjected to FLAG immunoprecipitations (IP), and the resulting complexes were blotted for the FLAG construct as well as endogenous p97. FLAG-Bap is tagged bacte- rial alkaline phosphatase and acts as a control. B, His6-tagged recombinant soluble p97 was incubated with the indicated proteins then precipitated and analyzed by Western blotting. C, ERAD assays were performed multiple times in HEK-293A cells for the indicated constructs and then quantified and graphed. Both SAKS1 truncations and UBXD3 are equivalent to the control at all time points but are statistically different from wild-type SAKS1 at the two later time points, p 0.01. CHX, cycloheximide.

Article Snippet: All point-mutated constructs were generated using the QuikChange site-directed mutagenesis kit (Stratagene). pQE9-His-p97 (wild type), ubiquitin-G76V-GFP, and pGEX hHR23A were obtained from Addgene (Addgene plasmids 14666, 11941, and 10864) (23–25).

Techniques: Transfection, Construct, Control, Recombinant, Incubation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The UBX Protein SAKS1 Negatively Regulates Endoplasmic Reticulum-associated Degradation and p97-dependent Degradation

doi: 10.1074/jbc.m110.158030

Figure Lengend Snippet: FIGURE 7. SAKS1 stabilizes NHK-HA at the ER and in the cytosol. A, HEK-293A cells were transfected with the indicated constructs and then separated into microsomal and cytosolic fractions 24 h later. Calnexin is an ER resident protein used as a marker. B, HEK-293A cells were co-transfected with His6-p97, NHK-HA, and either FLAG-SAKS1 or control DNA. The following day, the His6-p97 was precipitated, and the resulting complexes were analyzed by Western blotting for the indicated proteins.

Article Snippet: All point-mutated constructs were generated using the QuikChange site-directed mutagenesis kit (Stratagene). pQE9-His-p97 (wild type), ubiquitin-G76V-GFP, and pGEX hHR23A were obtained from Addgene (Addgene plasmids 14666, 11941, and 10864) (23–25).

Techniques: Transfection, Construct, Marker, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The UBX Protein SAKS1 Negatively Regulates Endoplasmic Reticulum-associated Degradation and p97-dependent Degradation

doi: 10.1074/jbc.m110.158030

Figure Lengend Snippet: FIGURE 9. SAKS1 protects polyubiquitin from deubiquitinases. A, overexpressed His6-p97 was precipitated from cells and then incubated with either BSA or recombinant SAKS1 at 37 °C. Samples were taken at the indicated time points and analyzed by Western blotting. B, Lys-48 (K48)-linked polyubiquitin chains were preincubated with either SAKS1 or BSA and then incubated overnight at 37 °C with recombinant ataxin-3. Samples were taken at time 0 and after overnight (O.N.) incubation and then blotted for the indicated proteins. C, cycloheximide (CHX) chase experiment was performed in HEK-293A cells co-transfected with the indicated tagged constructs, and lysates were collected at the indicated time points. The results of multiple experiments were quantified and graphed. The expression of ataxin-3 C14A significantly stabilizes substrate levels, although the wild-type ataxin-3 construct does not signifi- cantly decrease them. Co-transfection of wild-type ataxin-3 and SAKS1 does significantly decrease the stabilization of substrate otherwise seen with expres- sion of SAKS1 at later time points, p 0.05.

Article Snippet: All point-mutated constructs were generated using the QuikChange site-directed mutagenesis kit (Stratagene). pQE9-His-p97 (wild type), ubiquitin-G76V-GFP, and pGEX hHR23A were obtained from Addgene (Addgene plasmids 14666, 11941, and 10864) (23–25).

Techniques: Incubation, Recombinant, Western Blot, Transfection, Construct, Expressing, Cotransfection