Overexpression of catalytically active UBA1b is sufficient to restore polyubiquitination and promote TAK243 resistance in Uba1^M41L cells. (A) Schematic depiction of the complementary DNA (cDNA) constructs overexpressed in 32D Uba1^M41L cells. (B) Immunoblot for UBA1, V5, and ubiquitin in 32D Uba1^WT and Uba1^M41L parental cell lines and Uba1^M41L cells overexpressing UBA1 Δ1-40 (UBA1b), UBA1 Δ1-40/C632A (UBA1b/C632A), or luciferase (Luc). Immunoblot for VINC was performed as a protein loading control. (C) Proliferation of 32D Uba1^WT and Uba1^M41L parental cell lines and Uba1^M41L cells overexpressing UBA1b, UBA1b/C632A, or luciferase over 3 days in the presence of DMSO or a range of TAK243 concentrations. A single representative experiment (left, mean ± SD of 3 technical replicates) and calculated IC₅₀ values from 5 independent biological replicates (right, mean ± SD) are shown. Statistical significance was determined using a Mann-Whitney test. (D) Immunoblot for UBA1, V5, PARP1, H2AX, and H2AX pS139 proteins after 16-hour treatment of 32D Uba1^WT and Uba1^M41L parental cell lines and Uba1^M41L cells overexpressing UBA1b, UBA1b/C632A, or luciferase with DMSO or 10nM TAK243. Immunoblot for VINC was performed as a protein loading control.