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
![(A) Schematic representation of NoMi operation to isolate NoMi-expressing cells. A Flag tag on the outer surface of the cells can be used to select transduced NoMi-cells from cell mixture through antibody based magnetic bead affinity selection. A NoMi-containing single cell suspension is incubated with Flag tag affinity beads, whereby NoMi-cells get captured from the cell mixture by their Flag tag on the cell surface. After washing off non-NoMi expressing cells, bead-bound NoMi cells are cultured in a culturing flask. Following three days of culturing, cells are trypsinized and exposed to a magnet to ensure bead removal from the NoMi cells in the next culturing step. (B) Flag tag affinity bead composition dictates efficiency of NoMi expressing HEK293T cell capture. Representative fluorescent microscopy images of bead-captured NoMi cells at step 5 of the NoMi operation with <t>anti-Flag</t> <t>M2</t> magnetic beads that are 4% agarose beads [B1] and Flag antibody pre-coated Dynabeads [B2]. (C) The type of magnetic beads affects NoMi-cell selection. [C1] Quantification of copGFP fluorescence shows significantly more cells captured by anti-Flag M2 magnetic beads that are 4% agarose beads when compared to Flag antibody pre-coated Dynabeads. [C2] A similar tendency was verified upon mCherry fluorescence analysis. Data represents four replicates and are presented as mean with SEM (error bars) and *p < 0.01 Unpaired t -test. Scale bar is 100 μm in low and 25 μm in high magnification.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6823/pmc08886823/pmc08886823__nihms-1772419-f0002.jpg)
![(A) NoMi-Nanoluc detection in vivo with bioluminescence. [A1] Mice were intracranially injected into the striatum with NoMi-encoding lentiviral vectors (LV) to transduce local brain cells and generate a NoMi-EV source. Thirteen days after brain transduction, NoMi-Nanoluc was detected in the brains of living mice upon intraperitoneal injection of 100 μl of 2 times diluted Nano-Glo® In Vivo Substrate. [A2] NoMi-Nanoluc bioluminescent signal was stable over a timeframe of 6 min (N = 2 controls (injected with 1% PBS/BSA) and N = 2 NoMi (injected with LV encoding NoMi)). (B) Immunofluorescence of coronal sections of NoMi-injected mice. [B1] NoMi-expressing cells in striatum express copGFP, mCherry and anti-Flag tag. [B2] Percentage of NoMi transduced cells in striatum compared to non-transduced cells confirmed local expression of NoMi construct and thus NoMi-EV production. Scale bar 500 μm and 100 μm.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6823/pmc08886823/pmc08886823__nihms-1772419-f0005.jpg)
![(A) Generation of a stable NoMi-hNPCs culture with NoMi-lentivirus. [A1] Schematic representation of NoMi-hNPCs selection post-transduction with NoMi-lentivirus. [A2] Fluorescent copGFP and mCherry labels from stable transduced NoMi-expressing hNPCs selected with puromycin. Scale bar is 100 μm. (B) Isolation of NoMi-EVs from hNPCs media. [B1] NoMi-hNPCs secrete NoMi-EVs in the media that were pulled down with anti-Flag tag beads. [B2] copGFP-exRNA was detected in NoMi samples by ddPCR and their levels evaluated upon normalization with GAPDH. (N = 4). Data are presented as the mean with SEM (error bars) **p < 0.01 Unpaired t -test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6823/pmc08886823/pmc08886823__nihms-1772419-f0007.jpg)
