Extended Data Fig. 3. The IQ motif of SidJ is required for its optimal response to CaM.

a-b. The IQ motif is required for the optimal activity of SidJ in response to CaM. Serially diluted CaM were preincubated with SidJ (a) or the SidJ_IQ mutant (b) and SdeA in the glutamylation buffer at 37°C for 2 h. A cocktail containing 4xFlag-Rab33b, NAD⁺, and ubiquitin was added to the reactions. After incubation for another 2 h at 37°C. Proteins separated by SDS-PAGE were probed with the indicated antibodies. SidJ_IQ/DA, I841, Q842 were mutated to Asp and Ala, respectively. In each panel, data shown were one representative from at least three independent experiments that had similar results.

c. The SidJ_IQ mutant complements the intracellular growth defect of the ΔsidJ mutant. Acanthamoeba castellanii was infected with the indicated bacterial strains and intracellular bacteria were determined at the indicated time points. Each strain was done in triplicate and similar results were obtained in two independent experiments. Results are from one representative experiment done in triplicate from three independent experiments; error bars represent s.e.m. (n = 3).