Journal: bioRxiv

Article Title: Ubiquitin Ligase ITCH Regulates Life Cycle of SARS-CoV-2 Virus

doi: 10.1101/2024.12.04.624804

Figure Lengend Snippet: (A) Culture medium from vT2-WT and vT2-ITCH-KO cells expressing HA-tagged ubiquitin (Ub) and Flag-2×Strep tagged E was harvested for Strep AP. A decrease of the extracellular E protein induced by ITCH ablation was visualized by immunoblot (left) and dot blot analysis (right). (B) Lysates from HEK293 cells expressing Flag-tagged E with ITCH or ITCH-CS were subjected to Flag IP, followed by immunoblotting for autophagosome cargo receptors. E specifically precipitated p62 and ITCH promoted their interaction. (C-E) HEK293 cells were transfected with Flag-tagged E with ITCH or ITCH-CS. 24 h later, cells were analyzed by immunofluorescence with Flag, ITCH and p62 or LC3B or LAMP1 antibodies. ITCH enhanced the colocalization between E and p62 (C) or LC3B (D), while no change in the colocalization between E and LAMP1 (E) was noted. Scale bar, 10 μm. (F) Culture media and denatured lysates from control (CTRL) and p62 knock down (#1, #2) HEK293 cells expressing HA-tagged ubiquitin (Ub), Flag-tagged E were subjected to Flag IP (with incorporation of a washing step with urea before elution for culture media samples), followed by immunoblotting or dot blot analysis. p62 depletion resulted in the accumulation of intracellular E (both unmodified and ubiquitinated), while decreasing the level of extracellular E (n=3). (G, H) vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at 1 MOI for 10 h were subjected to immunofluorescence analysis with E and p62 or LC3B antibodies. ITCH-ablation decreased the colocalization between E and p62 (G) or LC3B (H). Scale bar, 10 μm. (I) A model of the function of ITCH in promoting autophagosome-mediated SARS-CoV-2 virion egress. ITCH-dependent ubiquitin modification enhances E binding with S and M binding with non-ubiquitinated E, resulting in the increase in virion formation and p62-dependent autophagosome targeting for release.

Article Snippet: The following primary antibodies were used: Flag (Sigma, F1804); Flag (Sigma, F3165); Flag (Cell Signaling Technology, 14793S); GAPDH (Invitrogen, MA5-27912); β-tubulin (Cell Signaling Technology, 2128S);s (BioLegend, 688102); Strep (Invitrogen, MA5-17283); CBD (New England BioLabs, E8034S); ubiquitin (Cell Signaling Technology, 58395S); K63-linkage-specific antibody (Enzo Life Sciences, BML-PW0600-0100); K48-linkage-specific antibody (Cell Signaling Technology, 8081S); Spike (Proteintech, 28867-1-AP); M (Proteintech, 28882-1-AP); E (Proteintech, 28904-1-AP); ITCH (Santa Cruz, sc-28367); ITCH (Novus Biologicals, NB100-68142); p62 (Cell Signaling Technology, 88588S and 7695S); GM130 (Proteintech, 11308-1-AP); LAMP1 (Cell Signaling Technology, 9091S); OPTN (Cayman Chemical, 100002); LC3 (Proteintech,14600-1-AP); LC3B (Cell Signaling Technology, 3868S); SARS-CoV-2 Membrane protein (Cell Signaling Technology, 15333S); SARS-CoV-2 Envelope protein (Cell Signaling Technology, 74698S); furin (Proteintech, 18413-1-AP); Cathepsin L (Proteintech, 10938-1-AP); NDP52 (Proteintech, 12229-1-AP); NBR1 (Proteintech, 16004-1-AP); FAM134B (Proteintech, 21537-1-AP); RTN3 (Proteintech, 12055-2-AP) and NIX (Proteintech, 12986-1-AP).

Techniques: Expressing, Western Blot, Dot Blot, Transfection, Immunofluorescence, Control, Knockdown, Infection, Modification, Binding Assay

Journal: The Journal of biological chemistry

Article Title: Liver-enriched transcription factor HNF-4 and ubiquitous factor NF-Y are critical for expression of blood coagulation factor X.

doi: 10.1074/jbc.271.4.2323

Figure Lengend Snippet: FIG. 3. Site 1 binds two distinct proteins and forms two DNA-protein complexes. A, sequences of the oligonucleotides used in Fig. 3 are shown. Only one strand is shown for each oligonucleotide. The numbering system uses translation start site as 11. B, an oligonucleotide spanning 268 to 239 of the Factor X gene (FXSite1) was labeled and incubated with 10 mg of nuclear extract (N.E.) from cell lines and rat tissues as indicated. Two DNA-protein complexes are designated complex A and complex B. The position of the free probe is marked as F. C, an oligonucleotide spanning site 1 was incubated with 12 mg of nuclear extract (N.E.) from human liver (lanes 1–12), 2 ml of in vitro translated HNF-4 in rabbit reticulocyte lysate (lane 13), or 2 ml of unprogrammed rabbit reticulocyte lysate (lane 14). Lanes 2–5 contain unlabeled site 1 oligonucleotide as cold competitors in 20 3, 100 3, 500 3, and 1000 3 molar excess. Lanes 7–10 contain unlabeled APF-1 (a strong HNF-4 binding site derived from 287 to 266 of apolipoprotein C III gene) as cold competitors in 20 3, 100 3, 500 3, and 1000 3 molar excess. Lane 12 contains 1 ml of HNF-4 antiserum. The positions of the antibody-HNF-4-DNA complexes are indicated by SS. D, probes containing the ACTTTG common motif from Factors VII, IX, X, and APF-1 were incubated with 12 mg of human liver nuclear extracts (N.E.) (lanes 1–8). One ml of HNF-4 antiserum was added in lanes 5–8. Positions of the immune complexes (supershift) are marked as SS. Two DNA-protein complexes specific to the APF-1 probe are marked by asterisks. E, the Factor X site 1 probe was incubated with 15 mg of nuclear extract (N.E.) from either HepG2 (lanes 1–3) or HeLa cells (lanes 4–6). In lanes 2 and 5, 200 3molar excess of unlabeled Sp1 consensus oligonucleotide was added. In lanes 3 and 6, 1 ml of Sp1 antibody was added. The position of a minor complex is denoted by an asterisk.

Article Snippet: Affinity-purified polyclonal antibody against amino acids 436–454 of Sp1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Labeling, Incubation, In Vitro, Binding Assay, Derivative Assay

Journal: The Journal of biological chemistry

Article Title: Liver-enriched transcription factor HNF-4 and ubiquitous factor NF-Y are critical for expression of blood coagulation factor X.

doi: 10.1074/jbc.271.4.2323

Figure Lengend Snippet: FIG. 4. Methylation interference analysis of the contact points of HNF-4 and Sp1 to site 1. A, On the left, Factor X site 1 was labeled on one strand (sense strand or antisense strand) and partially meth- ylated. The methylated probe was incubated with 100 mg of human liver nuclear extract and analyzed in regular electrophoretic mobility shift assay. The free probe fraction (F) and the complex B fraction (B, which contains HNF-4) were excised from the gel, cleaved with piperidine to reveal the G/A ladder, and resolved on a denaturing gel. On the right, the site 1 oligonucleotide was incubated with 10 footprint units of recombinant Sp1 and analyzed as described above. The free fraction (F) and bound fraction (B) were analyzed. The contact points revealed are marked as G. E indicates methylated positions that partially interfere with the binding. * indicates that methylation at these nucleotides enhances the protein binding. B, summary of results of methylation interference analysis at site 1.

Article Snippet: Affinity-purified polyclonal antibody against amino acids 436–454 of Sp1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Methylation, Labeling, Incubation, Electrophoretic Mobility Shift Assay, Recombinant, Binding Assay, Protein Binding

Journal: The Journal of biological chemistry

Article Title: Liver-enriched transcription factor HNF-4 and ubiquitous factor NF-Y are critical for expression of blood coagulation factor X.

doi: 10.1074/jbc.271.4.2323

Figure Lengend Snippet: FIG. 5. Increasing amounts of Sp1 diminish the binding of HNF-4 to site 1. Labeled Factor X site 1 oligonucleotide was incubated with 12 mg of human liver nuclear extracts (N.E.) in the presence of 0, 0.2, 1, and 2 footprint units (lanes 1–4) of recombinant Sp1.

Article Snippet: Affinity-purified polyclonal antibody against amino acids 436–454 of Sp1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Binding Assay, Labeling, Incubation, Recombinant

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 4. Effects of estradiol on cyclin protein expression. The experimental design was described in Fig. 2. Representative Western blots are shown for G1 phase cyclins (D1, D3, E) (A) and S/G2M phase cyclins (A, B1) (B). Controls for cyclin E are as described in Fig. 2.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 5. Estrogen induction of cyclin D1 and cyclin D3 mRNA expression. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, total cellular RNA was harvested. A, representative North- ern blots are shown for cyclin D1 and D3 mRNA from estradiol-treated and control cells. Arrows indicate the 4.5- and 1.5-kb cyclin D1 transcripts. B, graphical pres- entation of temporal changes in mRNA (open symbols) and protein (solid symbols, mean of two experiments) for cyclin D1 (left) and cyclin D3 (right).

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Expressing, Control

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 8. Activation of Cdk4 and Cdk2 following estrogen treatment. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were prepared and immunoprecipitated with antibodies to either Cdk4, cyclin E, or Cdk2, and then the kinase activity of the immunoprecipitates was determined by phosphorylation of GST-pRB773–923

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activation Assay, Control, Immunoprecipitation, Activity Assay, Phospho-proteomics

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 9. Composition of cyclin D1-as- sociated and cyclin E-associated complexes. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were prepared and immuno- precipitated with anti-cyclin D1 anti- serum or anti-cyclin E antibodies, and then these immunoprecipitates were sep- arated by SDS-PAGE and transferred to nitrocellulose membranes. A, cyclin D1 antiserum immunoprecipitates. The same filter was sequentially Western blotted for cyclin D1, Cdk4, p21, and p27. A rep- resentative blot is shown for each. B, rel- ative levels of cyclin D1 (E), Cdk4 (G), p21 (M), and p27 (f) were determined by den- sitometry and are expressed relative to the vehicle treated controls. Points repre- sent the mean of two separate experi- ments. C, cyclin E immunoprecipitates. The same filter was sequentially Western blotted for cyclin E, Cdk2, p21, and p27. A representative blot is shown for each. The asterisk marks the more mobile form of Cdk2 that is phosphorylated on Thr-160.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Control, SDS Page, Western Blot

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 10. Estrogen decreases inhibi- tory activity toward cyclin E-Cdk2. A, lysates were prepared from cells that were pretreated with antiestrogen and then treated with E2 (1) or vehicle (2). Active cyclin E-Cdk2 complexes that had been prepared from baculovirus-infected Sf9 cells were incubated with either lysis buffer only (labeled input) or with cell lysates. Lysates were also immunode- pleted with either anti-p27 antibodies, anti-p21 antibodies, or both and then in- cubated with recombinant cyclin E-Cdk2 complexes. Recombinant cyclin E-Cdk2 complexes were then recovered and as- sayed for histone (H1) kinase activity. The percentage of the input activity, de- fined as 100%, is also shown numerically below the autoradiograph. The same sam- ples were electrophoresed on a duplicate SDS-PAGE gel and then Western blotted for p21 and p27 to assess the binding of these proteins to recombinant cyclin E- Cdk2. B, the experiment described for panel A was repeated using either boiled lysis buffer (labeled input) or boiled ly- sates from cells treated with estradiol (1) or vehicle (2). C, MCF-7 cells were pre- treated with 10 nM ICI 182780 for 48 h and then treated with 100 nM estradiol or vehicle (control) at time 0. At intervals thereafter, whole cell lysates were pre- pared and assayed for cyclin E-Cdk2 in- hibitory activity as described for panel A.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activity Assay, Infection, Incubation, Lysis, Labeling, Recombinant, Autoradiography, SDS Page, Western Blot, Binding Assay, Control

Journal: The Journal of biological chemistry

Article Title: Estrogen-induced activation of Cdk4 and Cdk2 during G1-S phase progression is accompanied by increased cyclin D1 expression and decreased cyclin-dependent kinase inhibitor association with cyclin E-Cdk2.

doi: 10.1074/jbc.272.16.10882

Figure Lengend Snippet: FIG. 11. Cyclin E-Cdk2 activation is accompanied by loss of CDK inhibitor association and Cdk2 Thr-160 phosphorylation. MCF-7 cells were pretreated with 10 nM ICI 182780 for 48 h and then treated with 100 nM E2 or vehicle (control) at time 0. Whole cell lysates were prepared either 8 h after estrogen (8 h E2) or from control cells (ICI 182780). Lysates were then fractionated on a Superose 12 gel filtration column. A, fractions were precipitated with acetone and Western blot- ted for cyclin E (top panels) or assayed for cyclin E-Cdk2 histone (H1) kinase activity (bottom panel). The relative levels of cyclin E protein (E, G) and cyclin E-Cdk2 activity (M, f) in lysates from antiestrogen- pretreated cells and cells treated with estradiol for 8 h were determined by densitometry and are represented graphically. The elution of mark- ers of known molecular weight (ferritin, 440 kDa; catalase, 232 kDa; aldolase, 158 kDa) are indicated at the top of the graph. B, fractions 19 and 24 from the 8 h E2 lysate were immunoprecipitated with an anti- cyclin E antibody, and the immunoprecipitates were electrophoresed on a SDS-PAGE gel and transferred to a nitrocellulose filter. The same filter was then sequentially Western blotted for cyclin E, Cdk2, p21, and p27. A representative blot is shown for each. The asterisk marks the more mobile form of Cdk2 that is phosphorylated on Thr-160.

Article Snippet: Antibodies used were rabbit polyclonal antisera to human cyclin D1 (29), human cyclin E (C-19; Santa Cruz Biotechnology), and human Cdk2 (M2; Santa Cruz Biotechnology).

Techniques: Activation Assay, Phospho-proteomics, Control, Filtration, Western Blot, Activity Assay, Molecular Weight, Immunoprecipitation, SDS Page

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 2. Inhibition of p38 blocks NGF-induced neurite out- growth. A and B, PC12 cells were pretreated with the indicated con- centrations of SB203580 or 30 mM PD98059 for 30 min prior to treat- ment with 100 ng/ml NGF for 60 h. Representative images under a phase-contrast microscope (A) and quantitation of the percentage of cells with neurites (B) are shown. C and D, cells were cotransfected with pEGFP-C1 together with an empty expression vector SRa (2) or an expression vector encoding kinase-negative MKK6 (KN-MKK6), wild type p38 (WT-p38), or dominant-negative p38 (AGF-p38) (15). After 12 h the cells were treated with or without 10 mM SB203580. Then, 48 h after the transfection the cells were treated with or without 100 ng/ml NGF. Representative images of the transfected cells 60 h after NGF addition identified by the fluorescence of GFP (C) and quantitation of the percentage of cells with neurites (D) are shown.

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Inhibition, Microscopy, Quantitation Assay, Expressing, Plasmid Preparation, Dominant Negative Mutation, Transfection, Fluorescence

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 1. NGF induces p38 activation as well as ERK/MAPK ac- tivation. A, PC12 cells were treated with 100 ng/ml NGF or 50 mM arsenite for the indicated times (left) or with the indicated concentra- tions of NGF for 10 min (right), and the cell extracts were subjected to the immune complex kinase assay for p38 using activating transcrip- tion factor 2, ATF2, as a substrate (upper). The same cell extracts were subjected to immunoblotting with anti-phospho-p38 (middle) or anti- p38 antibodies (bottom). B, cells were pretreated with or without a p38 inhibitor SB203580 at 10 mM (lower or upper, respectively) for 30 min prior to NGF treatment as indicated, and the extracts were subjected to immunoblotting with anti-ERK/MAPK antibody. The electrophoreti- cally retarded bands represent active forms, i.e. phosphorylated forms of ERK/MAPK (ERK1 and ERK2, arrowheads) against inactive forms (arrows).

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Immune Complex Kinase Assay, Western Blot

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 3. Expression of a constitutively active MAPKK/MEK (SE- SE-KK) induces p38 activation as well as ERK/MAPK, and the p38 inhibitor blocks neurite outgrowth induced by SESE-KK. A, PC12 cells were cotransfected with pEGFP-C1 and either an empty expression vector SRa (2) or a constitutively active construct of MAPKK/MEK (SESE-KK (13); equivalent to Glu-217/Glu-221 MAPKK/ MEK in Ref. 7) expression vector and were treated with or without 10 mM SB203580. B, cells were cotransfected with HA-p38 or HA-ERK/ MAPK (MAPK) together with an empty expression vector SRa (2) or an expression vector encoding wild type MAPKK/MEK (WT-KK) or SE- SE-KK and assayed for the activity of HA-p38 or HA-MAPK. The activity of HA-p38 was also measured in cells treated with 100 ng/ml NGF for 10 min (1NGF). C, cells were cotransfected with pEGFP-C1 and either an empty expression vector SRa (2) or an SESE-KK expres- sion vector and were subjected to immunostaining with anti-phospho- p38 antibody.

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Expressing, Activation Assay, Plasmid Preparation, Construct, Activity Assay, Immunostaining

Journal: The Journal of biological chemistry

Article Title: Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells.

doi: 10.1074/jbc.273.38.24285

Figure Lengend Snippet: FIG. 4. EGF induces transient activation of p38 and, when combined with sustained activation of p38, causes neurite out- growth in PC12 cells. A, PC12 cells were treated with either 30 nM EGF (upper), 50 mM arsenite (upper), or 100 ng/ml NGF (lower) for the indicated times and assayed for p38 activity as described in the legend to Fig. 1A. B, cells were treated with EGF, NGF, or arsenite for the indicated times and subjected to immunostaining with anti-phospho- p38 antibody (lower; phase contrast, upper). C, cells were transfected with pEGFP-C1 together with either an empty expression vector SRa (2) or both wild type MKK6 and wild type p38 expression vectors (MKK6 & p38) and treated with or without 10 mM SB203580. 48 h after the transfection the cells were treated with or without EGF. Represent- ative images of the transfected cells 72 h after EGF addition identified by the fluorescence of GFP are shown. D, cells were treated with EGF, arsenite, or both for 1 h, washed, and then incubated in fresh medium. Representative images 60 h after the treatment under a phase-contrast microscope are shown.

Article Snippet: Anti-p38 antiserum was produced by immunizing rabbits with recombinant His-tagged p38.3 Anti-HA antibody and anti-p38 antibody were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Activity Assay, Immunostaining, Transfection, Expressing, Plasmid Preparation, Fluorescence, Incubation, Microscopy