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. 2018 May 22;9:1091. doi: 10.3389/fimmu.2018.01091

Figure 3.

Figure 3

Knockdown of heat shock protein 70 (Hsp70) by RNA interference (RNAi) reduces the tolerance of brine shrimp larvae to Vibrio parahaemolyticus infection. (A) Hsp70 protects brine shrimp larvae against V. parahaemolyticus MO904. Brine shrimp larvae obtained from females injected with either gfp (+Hsp70) or hsp70 dsRNA (−Hsp70) were pretreated with phloroglucinol (30 µM) for 2 h, rinsed to wash away the compound, and then allowed to recover for 2 h. The larvae were subsequently challenged with V. parahaemolyticus at 107 cells/ml. Phloroglucinol non-pretreated larvae with and without hsp70 that were challenged with V. parahaemolyticus served as positive control. Error bars represent the standard error of six replicates; asterisks represent significant difference between the control and the treatment, *P < 0.001. (B) Knockdown of hsp70 mRNA in brine shrimp larvae. Equal amounts of RNA from brine shrimp larvae were amplified by RT-PCR and the products were resolved by electrophoresis in agarose gels. M—100 bp DNA ladder, Lane 1—wild-type brine shrimp larvae, Lane 2—brine shrimp larvae injected with dsRNA gfp, Lane 3—brine shrimp larvae injected with dsRNA hsp70, Lane 4—wild-type brine shrimp larvae pretreated with phloroglucinol, Lane 5—brine shrimp larvae injected with dsRNA gfp and pretreated with phloroglucinol, Lane 6—brine shrimp larvae injected with dsRNA hsp70 and pretreated with phloroglucinol. (C) Knockdown of Hsp70 protein in brine shrimp larvae. The brine shrimp larvae from females injected with dsRNA for either gfp or hsp70 were resolved by electrophoresis in SDS polyacrylamide gels, blotted to nitrocellulose, and probed with Hsp70 antibody (3A3). The secondary antibody was HRP-conjugated donkey anti-mouse IgG. M—Protein ladder in kilodaltons, Lanes 1–3—wild-type brine shrimp larvae, Lanes 4–6—wild-type brine shrimp larvae pretreated with phloroglucinol, Lanes 7–9—brine shrimp larvae injected with dsRNA gfp and pretreated with phloroglucinol, Lanes 10–12—brine shrimp larvae injected with dsRNA hsp70 and pretreated with phloroglucinol.