Journal: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 2. The C-terminal region 99– 192 of HCV E1 protein determines lo- calization in the ER of the reporter CD8. FRT cells stably expressing CD8 (a–b) or CD8-E199–192 (c–f) were analyzed by double indirect immunofluorescence microscopy as detailed under “Experi- mental Procedures” and “Results.” a, c, e, and f, permeabilized cells; b and d, not permeabilized cells. a, c, and e, anti-CD8 mAb OKT8; b and d, polyclonal antibody anti-CD8; f, polyclonal antibody anti- SSRa; a, c, and e, Texas Red-conjugated anti-mouse secondary antibody; b, d, and f, fluorescein-conjugated anti-rabbit sec- ondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Stable Transfection, Expressing, Immunofluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 6. The juxtamembrane region 99–142 of the ectodomain of HCV E1 protein contains another determi- nant for ER localization. FRT cells sta- bly expressing CD8-E199–142 (a–d) and CD8-SV (e–f) were analyzed by double in- direct immunofluorescence microscopy as detailed under “Experimental Proce- dures” and “Results.” a, c–e, permeabi- lized cells; b and f, non-permeabilized cells. a, c, and e, anti-CD8 OKT8 mAb; b and f, polyclonal antibody anti-CD8; d, polyclonal antibody anti-calnexin; a, c, and e, Texas Red-conjugated anti-mouse secondary antibody; b, d, and f, fluores- cein-conjugated anti-rabbit secondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Expressing, Immunofluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 5. The TMD region of HCV E1 protein determines localization in the ER of the reporter CD8. FRT cells stably expressing CD8-E1155–192 were analyzed by double indirect immunofluorescence microscopy as detailed under “Experimental Procedures” and in the Results. a, c, and d, permeabilized cells; b, non-permeabilized cells. a and c, anti-CD8 mAb OKT8; b, polyclonal antibody anti-CD8; d, polyclonal antibody anti-calnexin; a and c, Texas red-conjugated anti-mouse secondary antibody; b and d, fluorescein-conjugated anti-rabbit secondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Stable Transfection, Expressing, Immunofluorescence, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 9. CD8-E199–142M protein is ex- pressed on the plasma membrane. HuH-7 cells were transfected with plas- mids expressing CD8-SV (a and b), CD8- E199–142 (c and d), and CD8-E199–142M (e and f) proteins. 36 h post-transfection, the cells were analyzed by single (a and b) and double (c and f) indirect immunoflu- orescence microscopy as detailed under “Experimental Procedures.” a, c, and e, permeabilized cells; b, d, and f, not per- meabilized cells; a–c and e, anti-CD8 mAb OKT8, Texas Red-conjugated anti-mouse secondary antibody; d and f, anti-CD8 polyclonal antibody, fluorescein-conju- gated anti-rabbit secondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Clinical Proteomics, Membrane, Transfection, Expressing, Microscopy

Journal: Journal of Biological Chemistry

Article Title: A New Determinant of Endoplasmic Reticulum Localization Is Contained in the Juxtamembrane Region of the Ectodomain of Hepatitis C Virus Glycoprotein E1

doi: 10.1074/jbc.m910400199

Figure Lengend Snippet: FIG. 12. The juxtamembrane region 99–142 of the ectodomain of HCV E1 determines intracellular localization of the AP protein. HuH-7 cells were transfected with plasmids expressing AP8 (a and b), AP8-SV (c and d), AP8- E199–142 (e and f), and AP8-E199–142M (g and h) proteins. 36 h post-transfection, the cells were analyzed by double indirect immunofluorescence microscopy as de- tailed under “Experimental Procedures.” a, c, e, and g, permeabilized cells, anti-AP mAb, Texas red-conjugated anti-mouse secondary antibody; b, d, f, and h, not permeabilized cells, anti-AP polyclonal antibody, fluorescein-conjugated anti- rabbit secondary antibody.

Article Snippet: The following antibodies were used: mouse mAb OKT8 (anti-CD8 protein), mouse mAb N1 (anti-CD8 protein), rabbit polyclonal anti-CD8 and rabbit polyclonal anti-SSra (16); mouse mAb G10-1 (anti-CD8 protein) (24)2; rabbit polyclonal anti-calnexin (17); rabbit polyclonal anti-calreticulin (Stress-Gene, Canada); rabbit polyclonal anti-alkaline phosphatase (Rockland, Gilbertsville, PA); mouse mAb anti-placental alkaline phosphatase (Chemicon International, Inc., Temecula, CA); peroxidaseconjugated anti-mouse and anti-rabbit IgG (Sigma-Aldrich); Texas Redconjugated anti-mouse IgG and fluorescein-conjugated anti-rabbit IgG (Jackson Immunoresearch Lab, West Grove, PA).

Techniques: Transfection, Expressing, Immunofluorescence, Microscopy

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 1 NAT10 is a novel substrate of PARylation in response to DNA damage. A, B MCF-7 and BT549 cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with or without 1 mM MMS for another 2 h or 6 Gy IR. Cells were harvested for IP and immunoblotting analyses with the indicated antibodies. C MCF-7 and BT549 cells were treated with or without 1 mM MMS for 2 h and subjected to IP analysis with an anti-NAT10 antibody. A total of 1% SDS was added to lysis buffer to remove all non-covalent binding. The immunoprecipitate was spotted onto a nitrocellulose membrane and the membrane was then examined using an anti-PAR antibody. Immunoblotting analysis was performed with anti-NAT10, PAR, and vinculin antibodies. D In vitro biotin pull-down assays were carried out by incubating purified GST-NAT10 or GST-CHFR with purified PAR (biotin-PAR polymer). GST-CHFR was used as a positive control

Article Snippet: Expression vectors, plasmid transfection, and lentiviral infection Myc-DDK-tagged MORC2 (Origene, #RC200518), Flag-His-NAT10 (Vigene, #CH874058), and GFPtagged NAT10 (Origene, #RG207082) cDNAs have been described previously [16].

Techniques: Western Blot, Lysis, Binding Assay, Membrane, In Vitro, Purification, Polymer, Positive Control

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 2 PARP1 PARylates NAT10 at K1016, K1017, and K1020 both in vitro and in vivo. A Four GST-NAT10 deletion mutants (∆1–201, ∆202–488, ∆489–753, and ∆754–1025) and GST control were bacterially purified, and subjected to in vitro PARation assay in the presence of PARP1 and NAD+. The reaction samples were resolved by SDS-PAGE, and analyzed by immunoblotting analyses with anti-PAR and anti-GST antibodies. B In vitro PARylation assays were performed using purified GST-NAT10 deletion fragments in the presence or absence of recombinant PARP1 enzyme, NAD+, and Olaparib. PARylated NAT10 was detected with an anti-PAR antibody. C–E GST-NAT10 deletion fragments were subjected to in vitro PARation assay as described in A. F, G Purified GST-NAT10 754–1025 proteins (WT, K1016A, K1017A, D1018A, and K1020A, K3A) were subjected to in vitro PARation assays in the presence of PARP1 and NAD+. PARylation of NAT10 was detected by immunoblotting with an anti-PAR antibody. In G, K3A represents the combined mutation in all three residues (K1016, K1017, and K1020). H Alignment of the NAT10 protein sequence among different organisms. Asterisk (*) indicates the full conservation of the residues of NAT10 among different species. I, J MCF-7 cells were transfected with HA-NAT10 or HA-NAT10 K3A expression vector. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h (I) or 6 Gy IR (J). Thereafter, IP and immunoblotting analyses were conducted with the indicated antibodies

Article Snippet: Expression vectors, plasmid transfection, and lentiviral infection Myc-DDK-tagged MORC2 (Origene, #RC200518), Flag-His-NAT10 (Vigene, #CH874058), and GFPtagged NAT10 (Origene, #RG207082) cDNAs have been described previously [16].

Techniques: In Vitro, In Vivo, Control, Purification, SDS Page, Western Blot, Recombinant, Mutagenesis, Sequencing, Transfection, Expressing, Plasmid Preparation

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 3 PARylation of NAT10 by PARP1 regulates its nucleoplasmic translocation and co-localization with MORC2. A MCF-7 cells were transfected with plasmid DNAs encoding Flag-MORC2, HA-NAT10, or HA-NAT10 K3A. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h or 6 Gy IR. IF staining was performed with an anti-Flag (green) or anti-HA (red) antibody. DNA was counterstained with DAPI (blue). Scale bar, 2.5 μm. The quantitative results of cells with NAT10 nucleoplasmic translocation are presented in the right panel. **p < 0.01; NS, no significance. B, C MCF-7 cells were transfected with HA-NAT10 and Flag-MORC2. After 48 h of transfection, cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with or without 1 mM MMS for another 2 h (B) or 6 Gy IR (C). IF staining was performed with an anti-Flag (green) or anti-HA (red) antibody. DNA was counterstained with DAPI (blue). Scale bar, 2.5 μm. The quantitative results of cells with NAT10 nucleoplasmic translocation are displayed in the right panel. **p < 0.01; ***p < 0.001. D, E PARP1-KO MCF-7 cells were transfected with HA-NAT10 and Flag-MORC2. After 48 h of transfection, cells were treated with or without 1 mM MMS for another 2 h (D) or 6 Gy IR (E). IF staining was performed with an anti-Flag (green) or anti-HA (red) antibody. DNA was counterstained with DAPI (blue). Scale bar, 2.5 μm. The quantitative results of cells with NAT10 nucleoplasmic translocation are displayed in the right panel. **p < 0.01; ***p < 0.001. Arrows indicate the colocalization between MORC2 and NAT10

Article Snippet: Expression vectors, plasmid transfection, and lentiviral infection Myc-DDK-tagged MORC2 (Origene, #RC200518), Flag-His-NAT10 (Vigene, #CH874058), and GFPtagged NAT10 (Origene, #RG207082) cDNAs have been described previously [16].

Techniques: Translocation Assay, Transfection, Plasmid Preparation, Staining

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 4 PARylation of NAT10 by PARP1 regulates its interaction with MORC2. A HEK293T cells were transfected with the indicated expression vectors. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h and subjected to IP and immunoblotting analyses with the indicated antibodies. B, C BT549 cells were transfected with plasmid DNAs encoding pCDH, HA-NAT10, or HA-NAT10 K3A. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h (B) or 6 Gy IR (C). IP and immunoblotting analyses were performed with the indicated antibodies. D–F MCF-7 and BT549 cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with or without 1 mM MMS for another 2 h. The sequential IP and immunoblotting analyses were performed with the indicated antibodies. G–I MCF-7 and BT549 cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with or without 6 Gy IR. The sequential IP and immunoblotting analyses were performed with the indicated antibodies

Article Snippet: Expression vectors, plasmid transfection, and lentiviral infection Myc-DDK-tagged MORC2 (Origene, #RC200518), Flag-His-NAT10 (Vigene, #CH874058), and GFPtagged NAT10 (Origene, #RG207082) cDNAs have been described previously [16].

Techniques: Transfection, Expressing, Western Blot, Plasmid Preparation

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 5 PARylation of NAT10 by PARP1 regulates MORC2 acetylation in response to DNA damage. A, B NAT10-KO MCF-7 and BT549 cells were transfected with plasmid DNAs encoding pCDH, HA-NAT10, or HA-NAT10 K3A. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h (A) or 6 Gy IR (B), and then subjected to IP and immunoblotting with the indicated antibodies

Article Snippet: Expression vectors, plasmid transfection, and lentiviral infection Myc-DDK-tagged MORC2 (Origene, #RC200518), Flag-His-NAT10 (Vigene, #CH874058), and GFPtagged NAT10 (Origene, #RG207082) cDNAs have been described previously [16].

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 7 PARylation of NAT10 by PARP1 is required for cell survival in response to DNA damage. A, B NAT10-KO MCF-7 and BT549 cells stably expressing pCDH, HA-NAT10 WT, or HA-NAT10 K3A were treated with increasing doses of MMS and then subjected to clonogenic survival assays. Representative images of survival colonies are displayed in A and the corresponding quantitative results are shown in B. C, D NAT10-KO MCF-7 and BT549 cells stably expressing pCDH, HA-NAT10 WT, or HA-NAT10 K3A were treated with or without 6 Gy IR, and then subjected to clonogenic survival assays. Representative images of survival colonies are displayed in C and the corresponding quantitative results are shown in D

Article Snippet: Expression vectors, plasmid transfection, and lentiviral infection Myc-DDK-tagged MORC2 (Origene, #RC200518), Flag-His-NAT10 (Vigene, #CH874058), and GFPtagged NAT10 (Origene, #RG207082) cDNAs have been described previously [16].

Techniques: Stable Transfection, Expressing

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 8 The proposed working model. Activated PARP1 after DNA damage catalyzes the PARylation of NAT10, which is required for the translocation of NAT10 from the nucleolus to the nucleoplasm. NAT10 relocalization increases its co-localization and interaction with its substrate, MORC2, thereby enhancing MORC2 K767Ac in response to DNA damage

Article Snippet: Expression vectors, plasmid transfection, and lentiviral infection Myc-DDK-tagged MORC2 (Origene, #RC200518), Flag-His-NAT10 (Vigene, #CH874058), and GFPtagged NAT10 (Origene, #RG207082) cDNAs have been described previously [16].

Techniques: Translocation Assay