Fig. 5

NK1-mediated responses to SP were downregulated by NK2 in A549 cells. A A549 cells lacking either NK were reconstituted with exogenous NKs. After incubation with calcium 6 reagent, cells were treated with 0.1 nM SP or NKA and the calcium responses were measured with a FlexStation^® 3 microplate reader. Maximal increases are shown in the graph. **: p < 0.01 (NK1 alone vs. NK1 + NK2). B A549 cells lacking NK2 and reconstituted with NKs were starved for 24 h and treated with 0.1 nM SP or NKA. Cells were lysed at the designated time point and cell lysates were subjected to western blotting with anti-ERK or phospho-ERK antibodies. The graph shows the intensity of maximal phosphorylation normalized with ERK. *: p < 0.05, ***: p < 0.001 (NK1 alone vs. NK1 + NK2). C Migration of A549 cells toward 0.1 nM SP. Cells lacking NKs and reconstituted with NKs in pictures were added into upper wells of transwell migration chambers and 0.1 nM SP was added to the lower well. After 18 h of incubation, non-migrated cells in upper well were removed with a cotton swab and cells migrated to the lower surface of the transwell were stained with Diff-quick staining solution and counted under a microscope. **: p < 0.01 vs. not treated. #: p < 0.05 (wild type vs. NK2KO or NK2KO + NK1)