Based on the idea of injecting a negative sequence, the compensation principle of the EHCS is analyzed in detail.
In Always No attack, an attacker decreases its local observations by injecting a negative offset in every sensing slot.
To block Activin signals, we injected a dominant-negative Smad2 mutant (Smad2-P445H), wharborsrbors a point mutation in the MH2 domain that alters the structure and prevents C-terminal phosphorylation (Eppert et al., 1996).
To block BMP signals, we injected a dominant-negative Smad1 mutant (Smad1-AVA) in which the C-terminal phosphorylated serines (Ser-463/465) were mutated to alanines to prevent phosphorylation but maintain receptor and co-factor interactions (Nojima et al., 2010).
To test this, we simultaneously injected a dominant-negative form of Slug (dnSlug), which inhibits neural crest migration (LaBonne and Bronner-Fraser, 2000), along with β-galactosidase, serving as a tracer for injection, in one cell of the two-cell-stage embryo, and then stained for the AVNA markers Phox2a and Hand2 by in situ hybridisation.
An equal number of HEK293 cells (∼5×106) containing only pLuxAB were injected as a negative control in the same volume.
Under the fluorescent microscope, the micropipette was placed in the MCL, and Lucifer yellow was injected with a negative current (1 10 nA) for 10 20 minutes until the glomerular tufts of the mitral cell appeared brightly fluorescent.
Cells containing only pLuxAB were injected as a negative control to determine if the substrates supplied by the luxCDEfrp genes in the pLuxCDEfrp plasmid were capable of being scavenged from endogenously available stocks within the host in the presence of the luciferase dimer formed by the products of the luxAB genes on the pLuxAB plasmid.
In parallel, PBS was injected as a negative control.
Poly IC alone (50 μg) was injected as a negative control.
One could then compare the effects on spontaneous spiking of injecting a comparable negative current somatically in the absence of inhibition, or even inject a conductance if one wanted to get fancy.
