Figure 1.

Identification of a Negative Regulatory Element in the 3′ UTR of Mouse Bok

(A–E) (A) List of constructs generated to narrow down the region containing destabilizing elements in the mouse Bok transcript sequence. Western blot analysis of BOK levels in total protein extracts prepared from HEK293T cells transiently transfected with (B) Bok cDNA or Bok CDS (with or without N-terminal FLAG tag), (C) Bok CDS with or without its 5’ UTR and/or 3’ UTR, (D) Bok CDS combined with the full length or truncated version of its 3’ UTR, or (E) Bok CDS combined with its wild-type 3’ UTR or harboring discrete point mutations in a predicted AU-rich element (ARE) therein. Each immunoblot is representative of at least three independent experiments. Tubulin and/or EGFP were used to normalize the transfection efficiency and gel loading. The double band detected for BOK corresponds to the full-length protein and a shorter version translated from a cryptic start codon at methionine 15 (Schulman et al., 2016). Bar graphs to the right of panels D and E show the quantification of BOK levels from quantitative immunoblots (using near-infrared fluorochromes) and real-time quantitative PCR. The data are presented as mean ± SD, N = 3. Statistical analysis was performed using a one-way ANOVA followed by a Tukey post-hoc test in GraphPad Prism. Significance levels, p > 0.05^ns, p < 0.01**, p < 0.001***.