Extended Data Fig. 7 ∣. The inteins Npu DnaE and Mtu RecA are not CRBN substrates.

(a) Left: hydrolysis of cyclic imides Fmoc-GGGFcQ or Fmoc-GGGFcN in PBS at 37 °C. (n = 3 biologically independent samples, error bars, which fall within symbols, represent mean ± SD). Right: hydrolysis of C-terminal cyclic imides on GFP-FcQ or GFP-FcN in PBS at 37 °C (n = 3 biologically independent samples, error bars, which fall within symbols, represent mean ± SD). (b) Schematic of the intein splicing mechanism with the penultimate step in intein excision that generates a C-terminal aspartimide highlighted, and the two intein constructs before and after splicing. X = O or S, R = H or CH₃. (c) Analysis of expression and splicing of Npu DnaE in E. coli with and without a V5 tag inserted in the intein. (n = 1 biologically independent replicate) (d) In vitro ubiquitination of V5-tagged cell lysate generated in c (n = 3 biologically independent replicates, all shown). (e, f) Effect of MLN4924 or lenalidomide pretreatment on 4-hydroxytamoxifen (4-HT)-induced splicing of GFP with HA-tagged Mtu RecA intein in HEK293T cells. For both e and f, data shown are representative of 2 biologically independent replicates. For uncropped western blot images, see Supplementary Fig. 11.