Fig. 2.

Knock down of group 1 LEA proteins in cysts by RNAi. a AfrLEA1 and GFP cDNA were amplified and dsRNAs were synthesized using PCR products. Reaction products were resolved in 1.5 % agarose and visualized with SYBR®Safe. Lane 1 GeneRuler 100 bp DNA Ladder (Thermo Scientific), sizes indicated on left in base pairs; 2 AfrLEA1 PCR product; 3 AfrLEA1 dsRNA; 4 GFP PCR product; 5 GFP dsRNA. b RT-qPCR was performed in duplicate with RNA from 25 to 30 cysts to determine AfrLEA1 and α-tubulin transcript copy number. AfrLEA1 copy numbers were normalized against α-tubulin and averaged for 4 and 6 cyst samples obtained from females injected with either GFP or AfrLEA1 dsRNA. AfrLEA1 copy number in cysts from females injected with GFP dsRNA was set at 100. Error bars represent standard deviation. The asterisk indicates that the mean copy number of group 1 LEA protein transcripts is statistically different from the group 1 LEA protein transcript copy number in cysts from females injected with GFP dsRNA (p < 0.05). c Protein extracts of 50 to 75 cysts from females injected with GFP dsRNA (1, 2) and AfrLEA1 dsRNA (3, 4) were resolved in SDS polyacrylamide gels, transferred to nitrocellulose, and probed with rabbit anti-group 1 LEA protein antibody followed by HRP-conjugated goat anti-rabbit IgG antibody and chemiluminescence. Molecular mass in kDa is on the left. Lanes 1 and 3, extracts of first brood cysts; 2 and 4, extracts of second brood cysts