Fig. 4.

Schlafen 12 acts by modulating deubuiquitylase activity. (a) Caco-2 cells transfected with non-targeting siRNA5 (siNT5) or a combination of both USP14 (siUSP14) and UCHL5 (siUCHL5) siRNA followed by transduction with control (Ad-CMV) or SLFN12 (Ad-SLFN12) viruses for 72 hours, and SI expression was assessed (n=6,*p<0.05). (b,c) Caco-2 cells were transduced with Ad-CMV or Ad-SLFN12 for 72 hours and USP14 and UCHL5 expression was measured (n=6 each,*p<0.05). (d,e,f) Caco-2 cells were transfected with either nontargeting siRNA NT5 or combined siRNA to UCHL5 and USP15 along with either an empty vector adenovirus (Ad-V) or adenovirus encoding SLFN12 (AdSLFN12). Western blots were performed for sucraseisomaltase (d), USP14 (e), and UCHL5 (f). (g,h) Caco-2 cells were transduced with Ad-CMV or Ad-SERPB12 and USP14 and UCHL5 expression measured. (i) Caco-2 cell lysates were immunoprecipitated with monoclonal USP14 or monoclonal SerpinB12 antibodies and immunoblotted with polyclonal SerpinB12 antibody. We immunoprecipitated Serpin B12 directly as a control to confirm the correct apparent molecular weight of the co-precipitating Serpin B12. (j) Incubation with recombinant human Serpin B12 stimulated deubuiquitylase activity vs. USP14 alone (n=4,*p<0.05 to substrate (sub) or SERPB12 alone; # p<0.05 vs. USP14 alone). (k) Incubation with recombinant human Serpin B12 did not affect the UCHL5 deubuiquitylase activity vs. UCHL5 alone (n=4,*p<0.05 to substrate (Sub) or Serpin B12). All statistics are by two-sided t test with Bonferroni corrections for multiple comparisons.