Figure 4. Loss of reggie-2/flotillin-1 in zebrafish embryos confers resistance to CT.

(A) An identical approach was used as with Derlin-1 (Figure 3), except that the morpholino was specific to the zebrafish reggie-2a/flot1a and reggie-2b/flot1b homologs, and protein extracts were immunoblotted with an anti–human flotillin-1 mAb (mouse kidney extract is positive control). (B) Control and reggie-2/flotillin-1 morphants (16-hpf) were intoxicated with 5 nM CT and imaged at 3 dpf. Three phenotypic categories were defined: no toxicity, resistant, and full toxicity. Percentages indicate the fraction of the population that segregated into each category. Control, n = 48; reggie-2/flotillin-1: n = 51. (C) In single-blind experiments, observers scored populations of control and reggie-2/flotillin-1 morphants that were intoxicated with a range of CT doses into the 3 categories. The results were averaged and reported as the mean percentage of the population represented by each category for each condition (error bars show SD of observer’s assessments; number of embryos/condition are indicated). Results are representative of 3 independent experiments. (D) Uninjected (No inj), control, or reggie-2/flotillin-1 morphants (16-hpf) were exposed to 5 nM CTB for 48 hours and washed, and crude protein extracts (pools of 5 embryos/condition) were analyzed by immunoblot using flotillin-1, β-actin, and CTB antibodies. (E) Control or reggie-2/flotillin-1 morphants intoxicated with 0.5 nM CT at 16 hpf were followed to 6 dpf and grouped into the three indicated categories (data combined from 2 independent experiments).