Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 1. SARS-CoV-2 3CLpro is released from infected cells by cell lysis and secreted by an unconventional protein-secretion mechanism (A) Immunoblots of 3CLpro precipitated by 33% trichloroacetic acid (TCA) from the conditioned media or in lysates from mock-infected or A549-ACE2 cells 48 hpi with SARS-CoV-2 (MOI 4, n = 5, N = 2). stds, 3CLpro standards. Densitometry of 3CLpro relative to 10 ng 3CLpro standard. (B) 3CLpro, b-tubulin, and GAPDH immunoblots and densitometry of 3CLpro in 33% TCA-precipitated conditioned media or cell lysate from mock-infected or SARS-CoV-2-infected A549-ACE2 cells 48 hpi with MOI 0.1 or 1.0. Dotted line, intracellular 3CLpro/tubulin ratio in MOI 1.0 cell lysate (n = 3/group). (C) Immunoblots and densitometry of 3CLpro in 33% TCA-precipitated conditioned media compared to matched lysates from SARS-CoV-2-infected Vero76 cells 48 hpi (MOI 1) or mock-infected cells (n = 3/group). 3CLpro ppt, 10 ng of 3CLpro precipitated from cell-naive media as a control; std, 10 ng of 3CLpro standard. (D) Immunoblots of 3CLpro in conditioned media concentrated by ultrafiltration and cell lysates from HEK293 cells transfected with 3CLpro expression plasmid for 48 h (n = 8, N = 2). (E) LDH activity in conditioned medium collected from 3CLpro transfected HEK293 cells (48 h) compared with the maximum amount of LDH released from lysed HEK293 cells (n = 6, N = 2). Absorbance was measured at 490 nm with 680 nm correction. (F) Immunoblots and densitometry of 3CLpro in concentrated conditioned medium and cell lysates from HEK293 cells transiently transfected with plasmids encoding 3CLpro or catalytic inactive 3CLpro Cys145Ala for 48 h (n = 5, N = 2). (G) Immunoblots and densitometry of 3CLpro or IFN-l1 after 24 h culture ± Golgi inhibitor 2 mM monensin (n = 6, N = 2). (H) 3CLpro, GSDMD, and b-actin loading control immunoblots and GSDMD densitometry of lysates from mock-infected or SARS-CoV-2-infected A549-ACE2 cells 48 hpi, MOI 0.1 or 1.0 (n = 3/group). (I) Schematic of candidate mechanisms of 3CLpro release from SARS-CoV-2-infected cells. Antibodies: anti-3CLpro (1:1,000, in-house), anti-b-tubulin (1:2,000, clone: BT7R), anti-b-actin (1:2,000, ab8226), anti-GAPDH (1:2,000, clone: GA1R), anti- GSDMD N-terminal antibody (1:500, clone: W18029B), and anti-FLAG M2 (IFN-l1 in G) (1:1,000, F3165). Data are shown as mean ± SD; statistical analysis of two groups was performed by two-tailed unpaired t test, three or more groups by one-way ANOVA with Tukey’s post hoc test, with a significance threshold of p < 0.05.

Article Snippet: SARS-CoV-2 3CLpro expression and purification Recombinant proteins were expressed from DNA expression vectors for active 3CLpro and inactive 3CLpro Cys145Ala control with C-terminal 3xFLAG-Myc-6xHis tags (Addgene 177334, 177335) or C-terminal 6xHis tag, purified as described5 and stored at 70 C in 3CLpro assay buffer (150 mM NaCl, 2 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl, pH 7.2).

Techniques: Infection, Lysis, Western Blot, Control, Transfection, Expressing, Plasmid Preparation, Activity Assay, Two Tailed Test

Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 2. SARS-CoV-2 3CLpro cleaves GSDMD at LQ29Y30SS to block GSDMD pore formation, whereas caspase cleavage generates func- tional GSDMD pores that directly secrete 3CLpro and nucleocapsid protein (A) Schematic of 3CLpro cleavage sites (non-prime side [P] in blue, prime side [P0] in red) and the caspase cleavage site (violet) in human GSDMD. Y, scissile bond.

Article Snippet: SARS-CoV-2 3CLpro expression and purification Recombinant proteins were expressed from DNA expression vectors for active 3CLpro and inactive 3CLpro Cys145Ala control with C-terminal 3xFLAG-Myc-6xHis tags (Addgene 177334, 177335) or C-terminal 6xHis tag, purified as described5 and stored at 70 C in 3CLpro assay buffer (150 mM NaCl, 2 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl, pH 7.2).

Techniques: Blocking Assay

Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 3. SARS-CoV-2 3CLpro is secreted through caspase-activated GSDMD and GSDME pores and retains activity in serum to dampen platelet activation (A) Recombinant 3CLpro (2 mM) was incubated with or without recombinant GSDME (3 mg) for 2 h at 37C and resolved on 4%–12% reducing SDS-PAGE stained with Coomassie (N = 2). 3CLpro with C-terminal 33FLAG-Myc-63His tag was used. (legend continued on next page)

Article Snippet: SARS-CoV-2 3CLpro expression and purification Recombinant proteins were expressed from DNA expression vectors for active 3CLpro and inactive 3CLpro Cys145Ala control with C-terminal 3xFLAG-Myc-6xHis tags (Addgene 177334, 177335) or C-terminal 6xHis tag, purified as described5 and stored at 70 C in 3CLpro assay buffer (150 mM NaCl, 2 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl, pH 7.2).

Techniques: Activity Assay, Activation Assay, Recombinant, Incubation, SDS Page, Staining

Journal: Cell reports

Article Title: SARS-CoV-2 3CL pro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL pro .

doi: 10.1016/j.celrep.2024.115080

Figure Lengend Snippet: Figure 4. SARS-CoV-2 3CLpro cleaves and inactivates IFN-l1 (A) Coomassie-stained 4%–12% SDS-PAGE gel of recombinant human IFN-l1 (2 mg) incubated alone, with active 3CLpro, or with inactive Cys145Ala mutant (2.5 mM) for 20 h showing an 15-kDa cleavage fragment (band 5) (N = 12). Bands 1–4 were identified by Edman sequencing to be the mature N terminus of intact IFN-l1, thus indicating multiple post-translational modified proteoforms of IFN-l1. The 3CLpro-cleavage fragment also commenced with the mature N-terminal sequence (band 5), indicating C-terminal truncation. (B) Amino-terminal oriented mass spectrometry (ATOMS) identification of 3CLpro cleavage sites in IFN-l1 by MS/MS peak analysis of isotopically heavy [C13H3]2- dimethylated(*) neo-N-terminal peptides 130*ACIQPQPTAGPRPR and 155*EAPKKESAGCLEASVTFNLFR, which were identified only in 3CLpro-treated samples. (C) Time course of 3CLpro cleavage of peptides spanning the P6–P60 ILSQLQ129Y130ACIQPQ(YR) sequence of IFN-l1 (top) and a non-cleavable P1-Q129A peptide (bottom) incubated over 4 h at 37C (n = 2, N = 2). (D) Schematic of the human IFN-l1 sequence highlighting the mature N terminus (black), the non-prime P5–P1 sequence (blue), and the prime P10–P50 sequence (red) of both cleavage sites. (E) IFN-l1 ribbon diagram structural model color coded according to B-factor analysis (PDB: 3O6G).68 Helices A to E are labeled as are the first and last resolved N-terminal (S41) and C-terminal (L189) residues, respectively, and both cleavage sites at the P1-Q and P10-A/E.

Article Snippet: SARS-CoV-2 3CLpro expression and purification Recombinant proteins were expressed from DNA expression vectors for active 3CLpro and inactive 3CLpro Cys145Ala control with C-terminal 3xFLAG-Myc-6xHis tags (Addgene 177334, 177335) or C-terminal 6xHis tag, purified as described5 and stored at 70 C in 3CLpro assay buffer (150 mM NaCl, 2 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl, pH 7.2).

Techniques: Staining, SDS Page, Recombinant, Incubation, Mutagenesis, Sequencing, Mass Spectrometry, Tandem Mass Spectroscopy, Labeling

Journal: STAR Protocols

Article Title: Affinity Purification of Label-free Tubulins from Xenopus Egg Extracts

doi: 10.1016/j.xpro.2020.100151

Figure Lengend Snippet:

Article Snippet: pGEX-6P-1 Stu2 1-590 , , Addgene Plasmid #38314.

Techniques: Recombinant, Protease Inhibitor, Filtration, Plasmid Preparation

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