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. Author manuscript; available in PMC: 2018 Sep 5.
Published in final edited form as: Cell Physiol Biochem. 2018 Jul 25;48(3):1274–1290. doi: 10.1159/000492019

Fig. 3.

Fig. 3.

Schlafen 12 acts through SerpinB12. (a) Lysates of Caco-2 cells transfected with empty vector (GST-alone) or SLFN12 (GST-SLFN12) were purified on GST columns. (b) Coprecipitation with antibody to GFP of SerpinB12 protein from Caco-2 cells overexpressing empty vector (pEGFP-C1), SLFN12 (pEGFP-C1-SLFN12), or a SLFN12 point mutant (pEGFP-C1-SLFN12, D233Q). Constructs and lysates were immunoprecipitated with monoclonal anti-GFP and immunoblotted with polyclonal anti-SerpinB12 (n=4,*p<0.05 by ANOVA followed by two-sided t test with Bonferroni correction). (c) Point mutations to the atypical ATP-binding region of SLFN12 prevent induction of sucrase-isomaltase (SI) promoter activity. Caco-2 cells were co-transfected with human SI-promoter reporter constructs without or with empty vector (pEGFP-C1) or SLFN12 wild-type construct (pEGFP-C1-SLFN12) or SLFN12 mutants (p-EGFP-C1-SLFN12 L222A, mutant1; p-EGFP-C1-SLFN12 D233A, mutant2; p-EGFP-C1-SLFN12 D233Q, mutant3; p-EGFP-C1-SLFN12 D233T, mutant4; and p-EGFP-C1-SLFN12 Y236F, mutant5) before luciferase reporter assay (n=6,*p<0.05 by ANOVA followed by two-sided t test with Bonferroni correction). (d) Caco-2 cells were infected with a lentivirus encoding either a V5 tag (as a control) or V5-tagged SLFN12, lysed, and immunoprecipitated with antibody to the V5 tag, prior to immunoblotting for Serpin B12 (n=3, *p<0.05), V5 (not seen in control lanes because the V5 tag alone is too small and runs off the gel), and Serpin B5, which was not observed to coprecipitate although we confirmed that the Serpin B5 antibody could be used for Western blotting in these cells (not shown). (e) Caco-2 cells were transfected with empty vector (pEGFP-C1) or SerpinB12 (pEGFP-C1-SerpinB12) constructs for 72 hours. Lysates were immunoprecipitated with monoclonal anti-SerpinB12 and immunoblotted with polyclonal anti-SLFN12. (f) Flow cytometry correlating endogenous Caco-2 SerpinB12 and SI expression. (g) Caco-2 cells were co-transfected with human SI-promoter reporter construct without or with empty vector (pEGFP-C1) or SerpinB12 (pEGFP-C1-SerpinB12) and luciferase reporter activity was assayed (n=6,*p<0.05). (h) Caco-2 cells were transduced with AAV-SerpinB12 virus or empty control virus (AAV-ZsGreen) and SI mRNA was measured (n=6,*p<0.05). (i) Caco-2 cells were transduced with Ad-CMV or Ad-SerpinB12 and western blot for SI protein was performed (n=6,*p<0.05 vs empty control virus). (j) Caco-2 cells were incubated with siNT or SerpinB12 siRNA (siSerpinB12), transduced with Ad-CMV or Ad-SLFN12 for 72 hours, and SI mRNA or (k) SI protein was measured (n=6, #p<0.05, to respective siNT; *p<0.05 siNT1-Ad-SLFN12 compared to siNT-Ad-V). All statistics are by two-sided t test with Bonferroni correction, with ANOVA performed as indicated.