Journal: PLoS ONE

Article Title: Ubiquitylation of Terminal Deoxynucleotidyltransferase Inhibits Its Activity

doi: 10.1371/journal.pone.0039511

Figure Lengend Snippet: (A) Endogenous TdT ubiquitylation in Jurkat cells. Immunoprecipitation was carried out using an anti-TdT antibody (lanes 4 and 6) or rabbit pre-immune serum (pre-IS) (lanes 3 and 5). Immunoprecipitants were subjected to SDS-PAGE and immunoblotted with anti-Ub (FK2) or anti-TdT antibody. One mg of whole cell lysate was used for each immunoprecipitation. (B) In vitro TdT ubiquitylation. The substrate His-TdT was incubated under ubiquitylation conditions with purified proteins. His-TdT and His-BPOZ-2 were purified from E. coli and the Flag-tagged proteins were purified from 293 T cells. To obtain Cul3/Rbx1 or BPOZ-2/Cul3/Rbx1 complexes, 293 T cells were co-transfected with the circled plasmids (lanes 10 and 11). After electrophoresis with a denaturing 7.5% polyacrylamide gel, ubiquitylated TdT was detected using an anti-TdT antibody. (C and D) Kinetics of TdT ubiquitylation in the absence (C) or presence (D) of the BPOZ-2/Cul3/Rbx1 complex. The incubation time after the addition of His-TdT is given above. The reaction products were separated by 7.5% SDS-PAGE and immunoblots were probed with anti-TdT antibody or anti-Flag antibody. The ratio of ubiquitylated His-TdT to unmodified His-TdT was determined with ImageJ. The band density for ubiquitylated His-TdT was defined as 100%.

Article Snippet: Mouse monoclonal antibodies (mAbs) against the Flag epitope tag (M2), actin (AC40), and ubiquitin (6C1) were obtained from Sigma; mouse mAbs against mono- and poly-ubiquitylated conjugates (FK2) from BIOMOL; mouse mAbs against the Myc epitope tag (4A6) from Upstate; rabbit pAb against the GST epitope tag from Affinity BioReagents; rabbit pAb against UbcH5, UbcH6, and UbcH7 from Boston Biochem; mouse mAb against penta-His from QIAGEN; and secondary anti-mouse and anti-rabbit IgG antibodies conjugated with HRP from New England Biolabs.

Techniques: Immunoprecipitation, SDS Page, In Vitro, Incubation, Purification, Transfection, Electrophoresis, Western Blot

Journal: PLoS ONE

Article Title: Ubiquitylation of Terminal Deoxynucleotidyltransferase Inhibits Its Activity

doi: 10.1371/journal.pone.0039511

Figure Lengend Snippet: (A) Immunoprecipitation using Myc-TdT as the bait. 293 T cells were co-transfected with expression vectors encoding Flag-UbcH5a (lanes 1 and 3) and Myc-TdT (lanes 2 and 3). Immunoprecipitation was carried out using an anti-Myc antibody. Immunoprecipitants were subjected to SDS-PAGE and following immunoblotting using an anti-Flag or anti-Myc antibody. (B) Immunoprecipitation using Flag-TdT as the bait. 293 T cells were co-transfected with expression vectors encoding Myc-UbcH6 (lanes 1 and 3) and Flag-TdT (lanes 2 and 3), and immunoprecipitation was carried out using an anti-Flag antibody. Immunoprecipitants were separated by SDS-PAGE and immunoblotted with an anti-Flag or anti-Myc antibody. (C) Immunoprecipitation using Flag-TdT as the bait. 293 T cells were co-transfected with expression vectors encoding Myc-UbcH7 (lanes 1 and 3) and Flag-TdT (lanes 2 and 3). Immunoprecipitation using an anti-Flag antibody. Immunoprecipitants were separated by SDS-PAGE and immunoblotted with an anti-Flag or anti-Myc antibody. (D) Immunoprecipitation of endogenous TdT in Jurkat cell lysates, using an anti-TdT antibody (lane 2) or rabbit pre-immune serum (pre IS) (lane 3). Immunoprecipitants were separated by SDS-PAGE and immunoblotted with an anti-UbcH5, anti-UbcH6 or anti-TdT antibody. Each immunoprecipitation used 1 mg whole cell lysate.

Article Snippet: Mouse monoclonal antibodies (mAbs) against the Flag epitope tag (M2), actin (AC40), and ubiquitin (6C1) were obtained from Sigma; mouse mAbs against mono- and poly-ubiquitylated conjugates (FK2) from BIOMOL; mouse mAbs against the Myc epitope tag (4A6) from Upstate; rabbit pAb against the GST epitope tag from Affinity BioReagents; rabbit pAb against UbcH5, UbcH6, and UbcH7 from Boston Biochem; mouse mAb against penta-His from QIAGEN; and secondary anti-mouse and anti-rabbit IgG antibodies conjugated with HRP from New England Biolabs.

Techniques: Immunoprecipitation, Transfection, Expressing, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Ubiquitylation of Terminal Deoxynucleotidyltransferase Inhibits Its Activity

doi: 10.1371/journal.pone.0039511

Figure Lengend Snippet: (A) UbcH5a enhances TdT ubiquitylation in 293 T cells. 293 T cells were transfected with plasmids encoding His-Ub (lanes 1, 3 to 5), Myc-TdT (lanes 2 to 5), and/or Flag-UbcH5a (lanes 1, 2, 4 and 5) in the indicated combinations. After a 24 h incubation, the cells were treated with 10 µM MG132 for another 12 h, lysed under denaturing conditions, and the ubiquitylated proteins were affinity-purified and separated by SDS-PAGE. Ubiquitylated Myc-TdT was detected by immunoblotting with an anti-Myc antibody. Myc-TdT and Flag-UbcH5a in the lysate were detected using an anti-Myc or anti-Flag antibody. (B) UbcH6 enhances TdT ubiquitylation in 293 T cells. 293 T cells were transfected with plasmids encoding His-Ub (lanes 1, 3 to 5), Flag-TdT (lanes 2 to 5), and/or Myc-UbcH6 (lanes 1, 2, 4 and 5) in the indicated combinations. After incubation for 24 h, the cells were treated with 10 µM MG132 for another 12 h. The cells were lysed under denaturing conditions, and the ubiquitylated proteins were affinity-purified and separated by SDS-PAGE. Ubiquitylated TdT was detected by immunoblotting using an anti-Flag antibody. Flag-TdT and Myc-UbcH6 in the lysate were detected using an anti-Flag or anti-Myc antibody. (C) UbcH7 does not enhance TdT ubiquitylation in 293 T cells. 293 T cells were transfected with plasmids encoding His-Ub (lanes 1, 3 to 5), Flag-TdT (lanes 2 to 5), and/or Myc-UbcH7 (lanes 1, 2, 4 and 5) in the indicated combinations. After a 24 h incubation, the cells were treated with 10 µM MG132 for another 12 h, lysed under denaturing conditions, and the ubiquitylated proteins were affinity-purified and separated by SDS-PAGE. Ubiquitylated TdT was detected by immunoblotting using an anti-Flag antibody. Flag-TdT and Myc-UbcH7 in the lysate were detected using an anti-Flag or anti-Myc antibody.

Article Snippet: Mouse monoclonal antibodies (mAbs) against the Flag epitope tag (M2), actin (AC40), and ubiquitin (6C1) were obtained from Sigma; mouse mAbs against mono- and poly-ubiquitylated conjugates (FK2) from BIOMOL; mouse mAbs against the Myc epitope tag (4A6) from Upstate; rabbit pAb against the GST epitope tag from Affinity BioReagents; rabbit pAb against UbcH5, UbcH6, and UbcH7 from Boston Biochem; mouse mAb against penta-His from QIAGEN; and secondary anti-mouse and anti-rabbit IgG antibodies conjugated with HRP from New England Biolabs.

Techniques: Transfection, Incubation, Affinity Purification, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Functionally Distinct Effects of the C-Terminal Regions of IKKε and TBK1 on Type I IFN Production

doi: 10.1371/journal.pone.0094999

Figure Lengend Snippet: 293ET cells were transfected with FLAG-tagged wt IKKε or mutants as indicated on top and were fixed at 20 h post-transfection. Fixed cells were stained with anti-FLAG and anti-IRF3 antibodies and were observed by confocal microscopy. Green, red and blue fluorescence in merged figures (top panels) indicate the endogenous IRF3, FLAG-tagged IKKε and nucleus, respectively. Single channel images are also shown in lower panels.

Article Snippet: Mouse monoclonal antibodies against FLAG M2 and α-tubulin and anti-FLAG agarose beads were purchased from Sigma (St Louis, MO).

Techniques: Transfection, Staining, Confocal Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: DNA Damage Stress and Inhibition of Jak2-V617F Cause Its Degradation and Synergistically Induce Apoptosis through Activation of GSK3β

doi: 10.1371/journal.pone.0027397

Figure Lengend Snippet: (A) UT7/Jak2-V617F cells, cultured without Epo, were treated for 7 h with or without 1 µM JakI-1, as indicated, with 1 µM etoposide (VP16) added for indicated times before harvest. Cells were then analyzed for activation of Bax or caspase-3 by flow cytometry. Percentages of cells with activated Bax or caspase-3 are plotted. FSC: forward scatter. (B) UT7/Jak2-V617F cells, cultured without Epo, were pretreated for 1 h with 1 µM JakI-1, 100 µM Boc-d-fmk, or 1 µM GSK3I-5, as indicated. Cells were further treated for 6 h with or without 5 µM VP16 and analyzed for loss of mitochondrial membrane potential and activation of Bax or caspase-3, as indicated, by flow cytometry. (C) 32D/EpoR cells were deprived of Epo and pretreated with 100 µM Boc-d-fmk (B-d-f), as indicated, or left untreated. Cells were then treated for 5 h with or without etoposide (VP16), as indicated, before harvest. Immunoprecipitates obtained using anti-Jak2 (06–255) were analyzed by immunoblotting using anti-Jak2 (M-126) and anti-Jak2 (C-20), as indicated. The numbers of cells treated with etoposide were 2.5 times that of cells untreated. An arrow indicates the 100-kDa band. (D) UT7/Jak2-V617F cells, cultured without Epo, were pretreated for 1 h with or without 1 µM JakI-1, as indicated. Cells were then treated for 8 h with or without 5 µM etoposide (VP16), as indicated, before harvest. Immunoprecipitates obtained using anti-Jak2 (06–255) were analyzed by immunoblotting using indicated antibodies. The number of cells treated with etoposide was 2 times that of cells untreated. (E) UT7/Jak2-V617F cells, cultured without Epo, were pretreated for 1 h with 1 µM JakI-1 and 20 µM Boc-d-fmk (B-d-f), as indicated. Cells were then treated for 6 h with or without 5 µM etoposide (VP16), as indicated, before harvest. Immunoprecipitates obtained using anti-Flag was were analyzed by immunoblotting using anti-Jak2 (06–255). The numbers of cells treated with etoposide were 2.5 times that of cells untreated.

Article Snippet: A mouse monoclonal antibody against FLAG (M2) and β-actin were purchased from Sigma.

Techniques: Cell Culture, Activation Assay, Flow Cytometry, Western Blot