Fig. 5
Identification of potential functional domains in T.gHB1 GRA15 for NF-κB promoter activation. a Illustration of full-length and truncated GRA15 proteins. Bioinformatics analysis was performed on the full length of GRA15 (T.gMe49), GRA15 (T.gHB1) and GRA15 (T.gRH). GRA15 (T.gHB1) and GRA15 (T.gRH) have a transmembrane region (the area marked in red). GRA15 (T.gMe49) has two transmembrane regions (areas highlighted in red) and lacks 518–602 AA regions compared to GRA15 (T.GHB1). Five truncated plasmids were constructed according to this design. b HEK293T cells were co-transfected with NF-κB luciferase reporter plasmid along with each of the GRA15-containing plasmids, and after 24 h, the luciferase activity was measured in cell lysates. Data are presented as the values with standard error of three independent experiments. c The FLAG-tagged GRA15-containing plasmid GRA151-518 (3) was transfected into HEK293T cells for 24 h, and p65 was stained by IFA. Hoechst, DNA-intercalating dye. d The percentage of cells expressing FLAG tag was calculated, and p65 positivity in the cell nucleus was assessed by co-staining with the FLAG-positive cells. Data were generated in three independent experiments (mean ± SD; n = 3). e HEK293T cells were transfected with different GRA15-containing plasmids as indicated for 30 h. TNF-α was used as positive control. Expression levels of IκBα and of phosphorylated IκBα were detected by Western blotting. GAPDH was used as a loading control. f HEK293T cells were co-transfected with NF-κB and pTL-TK luciferase reporter plasmid along with each of the GRA15-containing plasmids, and 18 h post, MG-132 was added and lasted for 6 h. After 24 h the luciferase activity was measured in cell lysates. Data are presented as the values with standard error of three independent experiments (***P < 0.001; ****P < 0.0001; NS not significant)