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. 2017 Feb 23;8(2):e2627. doi: 10.1038/cddis.2017.51

Figure 2.

Figure 2

Figure 2

ULK2 represses the NF-κB-inducing activity of CARMA2sh. (a) HEK293T cells were transiently cotransfected with expression vectors encoding for the indicated polypeptides, together with NF-κB-luciferase and β-galactosidase reporter vectors. About 24 h after transfection, cell lysates were prepared and luciferase activity was measured. Data were analyzed by Student's t-test, and a P-value ⩽0.05, indicated with an *, was considered significant. Data shown represent relative luciferase activity normalized against β-galactosidase activity and are representative of at least 10 independent experiments done in triplicate. Lower panel: a fraction of the cell lysate was analyzed by immunoblot assay to monitor protein expression. (b) NHEK were transfected with an expression vector encoding for ULK2 or GFP. About 24 h later, cells were left in PBS or exposed to the indicated heat-killed microorganisms for 16 h, and the expression level of selected NF-κB target genes was monitored by real-time PCR. Graphs show the fold changes respect to the GFP-transfected cells left in PBS. Data were analyzed by Student's t-test, and a P-value ⩽0.05, indicated with an *, was considered significant. Data shown are representative of at least three independent experiments done in triplicate. (c) NHEK cells were infected with lentiviral vectors encoding for shULK2 or a scramble control sequence, and then treated and assayed as in b. Graphs show the fold changes respect to the scramble-infected cells left in PBS. Data shown are representative of at least three independent experiments done in triplicate. Efficacy of ULK2 attenuation by shRNAs was monitored by real-time PCR and immunoblot assay. (d) NHEK were infected with lentiviral vectors encoding for indicated shRNAs or a scramble control sequence and then treated and assayed as in (c). (e) NHEK cells were left in PBS or exposed to heat-killed S. aureus cells for 16 h. Where indicated, the cell lysate was treated with CIP for 30 min at 37 °C and endogenous CARMA2sh expression was analyzed by immunoblot assay