mouse anti-flag epitope tag m2 mab  (Millipore)

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