Fig. 5: The HER2/p65 dephosphorylation (Ser536) axis negatively modulates miR-22 expression.

a Whole cell lysates prepared from S05 and 182^R-6 cells were subjected to western blot analysis using the indicated antibodies; actin served as a loading control. b 182^R-6 cells were transiently transfected with either 100 nM Neu siRNA (pool of 3–5 target-specific 19–25 nucleotide sequences in length) or control siRNA-A. At 72 h after transfection, whole cell lysates were prepared, and the western blot analysis was performed using the indicated antibodies, as described in the “Methods” section. Actin served as a loading control. c 182^R-6 cell were transfected with either 100 nM Neu siRNA or control siRNA-A. At 72 h after transfection, the total RNA was isolated, and qRT-PCR was performed using a primer set for hsa-miR-22. RNU6-2 was used as a reference gene to normalize the miR-22 expression. d Diagram of wild-type and mutant miR-22 promoter constructs. e HEK293 cells grown to 90% confluency were transiently transfected with either 50 or 100 ng reporter plasmid in combination with 0.5 μg GFP-RelA and 5 ng pRL-TK plasmid. At 24 h after transfection, the luciferase activity was measured as described in the “Methods” section. f, g 182^R-6 cells grown to 80% confluency were exposed to the indicated concentrations of the IKK2 inhibitor sc-514. 1 h after exposure, the cells were incubated with the indicated concentration of LPS. At 24 h after treatment, total RNA was isolated and subjected to qRT-PCR analysis for miR-22 (f), and nuclear lysates were prepared and subjected to western blot analysis p65 (g). Nuclear histone H3 served as a loading control. * indicates p < 0.05