Fig. 6.

Nuclear NDR1 promotes STAT1 translation. a−c RAW264.7 cells stably expressing flag-NDR1 or control transfectants (a), WT or NDR1^−/− PMs (b), NDR1 silencing PMs (c), were treated with metabolic labeling reagents (AHA). Subsequently corresponding cell lysates were labeled with biotin for nascent proteins via azide/alkyne reaction, followed by immunoprecipition with anti-STAT1 antibody or nonspecific IgG. d Immunoblot analysis of flag-tagged full-length NDR1 or NDR1 ΔNLS mutant expression in cytoplasmic and nuclear fractions from RAW264.7 cells stably expressing flag-NDR1 or flag-NDR1 ΔNLS mutant. e Immunoblot analysis of STAT1 expression in RAW264.7 cells stably overexpressing NDR1, NDR1 ΔNLS mutant or empty vector. f FACS analysis of RAW264.7 cells as in e infected with VSV-eGFP. g ChIP-qPCR analysis of the enrichment of NDR1 to miR146a intergenic region with anti-flag M2 magnetic beads in lysates of RAW264.7 cells stably expressing flag-NDR1 or control transfectants. h Biotin-labeled DNA was incubated with lysates of PMs for 30 min at 25 °C. Streptavidin sepharose beads were then added and incubated for another 30 min. The beads were washed and then resolved by 2× SDS loading buffer for immunoblotting. i Biotin-labeled DNA (miR146a intergenic region or control DNA) and purified myc-NDR1 protein were incubated in DNA pull-down buffer for 30 min at 25 °C. Streptavidin sepharose beads were then added and treated as in h for immunoblotting. Data are mean ± SD and are representative of three independent experiments. Student’s t test was used for statistical calculation. *p < 0.05, **p < 0.01, ***p < 0.001. See also Supplementary Fig. 6