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. 2017 Jun 15;66(7):2033–2043. doi: 10.2337/db16-1023

Figure 1.

Figure 1

Generation and characterization of Jurkat-derived clones deficient in or overexpressing UBASH3A. A: Two Jurkat-derived UBASH3A−/− clones, 2.1D6 and 2.1F7, were generated by CRISPR targeting exon 2 of the UBASH3A gene. Partial exon 2 sequences of parental Jurkat cells and the two UBASH3A−/− clones are shown. Insertions and deletions are underscored and italicized. B: Whole-cell lysates from 2.1D6, 2.1F7, and Jurkat cells were immunoprecipitated with either anti-UBASH3A (lanes 1–3) or IgG (lane 4). The immunoprecipitates were subjected to immunoblotting with a different anti-UBASH3A antibody. UBASH3A and its monoubiquitinated form are indicated by arrow and asterisk, respectively. C: Immunoblot analysis of whole-cell lysates from 2.1D6, 2.1F7, and Jurkat cells using anti-UBASH3B. D: Whole-cell lysates from three Jurkat-derived clones overexpressing UBASH3A with a COOH-terminal V5 tag were subjected to immunoblotting with anti-V5 and subsequently with anti–γ-tubulin after stripping. The blots in B–D are representative of two independent experiments. KO, knockout.