Fig 2: Mapping the interaction between USP21 and KCTD6–CUL3 E3-ligase complex. (A) USP21 interacts with KCTD6[114–237]. HEK293T cells were transfected with full-length (FL) or truncated KCTD6, GFP or GFP–USP21, and immunoprecipitations (IPs) were performed and analysed as indicated. IB, immunoblot. (B) The overall fold similarity between the favoured QUARK ab initio model of the KCTD6 Smr-like domain [SmrL (109–213)] and the C-terminal Smr domain of human NEDD4BP2 (PDB code 2VKC; Diercks et al., 2008). Each is coloured in a spectrum from blue (N-terminus) to red (C-terminus). A broken line indicates the boundary between the functionally dispensable C-terminal region [188–213] and the remainder. (C,D) HEK293T cells that had been transfected with FLAG–KCTD6 (C) or FLAG–CUL3 (D), and GFP-tagged USP21 fragments were lysed in RIPA buffer. Protein lysates were used for immunoprecipitation with an anti-GFP antibody. Asterisk indicates a monoubiquitylated form of FLAG–KCTD6. (E) Diagram summarising the interactions between USP21, KCTD6 and CUL3, components of the BTB–CUL3–Rbx1 E3-ligase complex. Residues 185–565 in USP21 encompass the necessary elements for the interaction with KCTD6 (residues 114–187). Cullins share a homologous C-terminus that interacts with the RING-finger protein Rbx1.

Fig 3: Indirect interaction of USP21 with cullin-3. (A) Diagram of the featured CUL3 constructs: full-length CUL3, a BTB-domain-binding mutant (CUL3m) and a dominant-negative C-terminal truncation mutant [CUL3(1–250)]. Asterisks indicate point mutations (see Materials and Methods). (B) USP21 interacts with CUL3 in HEK293T cells. HEK293T cells were transfected as indicated, and lysates were subjected to immunoprecipitation (IP) with anti-GFP and anti-FLAG antibodies as appropriate. Asterisk indicates a higher-molecular-mass form of FLAG–CUL3. (C,D) HEK293T cells were co-transfected with USP21 and with either CUL3m (C) or with CUL3(1-250) (D). Protein lysates were subjected to immunoprecipitation with anti-GFP or anti-FLAG antibodies. The interacting proteins were analysed by western blotting (IB) with anti-GFP and either anti-CUL3 or anti-FLAG antibodies as indicated: USP21(/CS), USP21 and USP21-C221S.

Fig 4: Reverse-transcription polymerase chain reaction of the spliced RNA. Reverse-transcription (RT) polymerase chain reaction (PCR) of the spliced RNA was performed. (A,B) PCR using a primer set (F4 and R4) flanking exon 9. Wild-type Cullin 3 (Cul3) cDNA produced a single product that included exon 9 (664 bp). If exon 9 of CUL3 was skipped, the expected PCR product would be 493 bp. We detected only the bands including exon 9. (C) Representative RT-PCR sequences. Complementary DNA with properly spliced junctions between exons 8 and 9 was confirmed in both types of knock-in mice. WT: wild type. G(−1)A: Cul3 c.1207 −1G>A knock-in mice. T(−6)G: Cul3 c.1207 −6T>G knock-in mice.

Fig 5: Blood Pressure, Heart Rate, Activity, and Circadian Rhythm. Four weeks after completion of tamoxifen injections, BP and activity were measured by radiotelemetry for 10 min each hour for 7 consecutive days in male control and E-CUL3∆9 mice. (A) Systolic blood pressure (SBP), (B) diastolic blood pressure (DBP), (C) HR, and (D) activity data were collapsed onto a single 24 h light/dark cycle. The time course data were analyzed using 2-way ANOVA with repeated measurements to determine the effect of genotype, time, and interaction. Averages of the 12-hour light phase (6AM-5PM) and 12-h dark phase were shown in dot plots. Despite marked circadian variations in all parameters, no effect of genotype was detected (P > 0.05, two-way ANOVA). The p values for main effects of two-way ANOVA were indicated in each panel. (E) Cosinor analysis of continuous 96-h SBP data that represented the 7-d telemetry recordings. The mean and SEM of SBP amplitude and PhaseShift generated by the cosinor analysis was tested by Student's t-test. Shaded areas indicate the 12-h dark phase (6PM–5AM or Zeitgeber time 0–12). Data were plotted as mean ± SEM. n = 9–11/genotype. *, P < 0.05.