We used this strat Axitinib cancer Inhibitors,Modulators,Libraries egy to investigate whether MEK D site mutations could alter ERK phosphorylation in the A. gambiae midgut. Specifically, pMEK2 and pMEK5 plasmids were microin jected into the hemocoels of vitellogenic female mosqui toes that had fed on blood 16 24 h earlier. Following injection, mosquitoes were allowed to oviposit and eggs were collected for rearing. To confirm Inhibitors,Modulators,Libraries midgut specific overexpression of the vari ant MEK alleles, 4 d old F0 adult female offspring of plasmid injected A. gambiae were allowed to feed on blood for 30 min. Age matched F0 offspring from mosqui toes from the same cohorts that were not injected with plasmid and, hence, not transformed were fed alongside transformed F0 females as controls to assess specificity of transcript detection.

At 2 h post feeding, midguts from six female A. gambiae in each group were dissected into PBS. TaqMan qRT PCR with probes specific to exogenous variant MEK alleles revealed no detectable signals from midguts or carcasses of Inhibitors,Modulators,Libraries non transformed F0 females. hence, relative levels Inhibitors,Modulators,Libraries of endogenous MEK mRNA in non transformed females were used for compari son to variant MEK allele expression in transformed F0 females. From replicated F0 cohorts, midgut expression of mRNAs encoding catalyt ically active MEK and catalytically active MEK K3MK6M were detected at levels four and three fold higher than endogenous MEK mRNA levels in non transformed mosquitoes. Surpris ingly, expression of exogenous variant MEK alleles in the carcass appeared to be equivalent to endogenous MEK mRNA levels in the same tissue in non transformed F0 females.

While variant MEK allele probes specif ically detected transcripts in transformed mosquitoes, we cannot exclude the possibility that some level of en dogenous MEK mRNA is also detected by these probes, which would explain signal detection in the carcass of transformed F0 females Inhibitors,Modulators,Libraries despite use of a midgut specific promoter. Midgut directed overexpression of MEK alleles with D site polymorphisms decreased ERK phosphorylation Based on midgut biased variant MEK mRNA expression in transformed F0 mosquitoes, we examined this tissue from transformed F0 females for relative levels of ERK phosphorylation. At 2 h post blood meal, 30 45 midguts from each group were dissected, pooled and processed for immunoblotting. As shown in Figure 5B, the response to pMEK2 transformation was correlated with the response to pMEK5 across replicates. In particular, 4 of 5 replicates showed a re duction in ERK phosphorylation levels in midgut epithelia of mosquitoes Idelalisib PI3K that overexpressed the catalytically active but docking deficient MEK relative to levels in mosquitoes that overexpressed catalytically active MEK.