Figure 3. SPRTN is a DNA binding protease.
(A) Western blot analysis of GFP-SPRTN expression. GFP-SPRTN and corresponding mutants were immunoprecipitated after 16 hr of transient transfection in 293T-HEK cells. Arrows indicate full-length (FL) or cleaved fragments. (B) Western blot analysis of histone H3 cleavage in cells overexpressing GFP-SPRTN, GFP-SPRTN-E112A or GFP-SPRTN-∆C. (C) EMSA of full-length recombinant SPRTN or SPRTN-∆C (a.a. 1–216) and 0.25 uM 6-FAM-39-mer single stranded oligodeoxynucleotide (6-FAM-ssODN) or (D) 0.25 uM 6’FAM-39-mer double stranded oligonucleotide (6-FAM-dsODN) .
Figure 3—figure supplement 1. SPRTN self-cleaves in vivo.
(A) Western blot analysis of SPRTN self-cleavage in cells. 293T-HEK cells were transfected with GFP-SPRTN or GFP-SPRTN-E112A. Experiment was performed in duplicate. Cells were directly lysed in 1X Sample buffer. Asterisk denotes a non-specific cross-reacting band that serves as a loading control. (B) Schematic illustration describing expression system to detect SPRTN self-cleavage in live cells. HeLa cells were transfected with a dual tag GFP (N-terminus) and mCherry (C-terminus) SPRTN expression plasmid.