1. Total hydrolytic enzyme activity and maturation of purified phagosomes.
Total hydrolytic enzyme activity and maturation of purified phagosomes. (A) The total content of the N-acetyl-glucosaminidase and N-acetyl-mannosidase lysosomal enzymes in wt, lmpA kd, lmpB ko and lmpC ko cells was measured with an ELISA microplate reader after incubating cell lysates with the respective chromogenic substrates. The activity of proteolytic enzymes was assessed with silica beads coupled to the self-quenching reporter DQ Green-BSA and the reference dye Alexa Fluor 594 in K-acetate buffer (pH 4). Data are mean±s.e.m. normalized to wt of three to four independent experiments performed in up to technical sextuplicates (ns, nonsignificant; *P≤0.05, **P≤0.01, two-tailed Student's t-test). (B–D) Phagosome maturation in wt and lmpA kd cells. Phagosomes containing latex beads were purified, and specific proteins were monitored by quantitative immunoblotting as described previously (Gotthard et al., 2006b). Representative blots of VatM, VatA, p80, LmpB or LmpC between phagosomes from wt and lmpA kd cells (B). Representative blots and the corresponding quantifications of two independent experiments for actin (C) and CprG (D), demonstrating differences in their maturation profiles in the lmpA mutant. Data are mean±s.e.m.
Natascha Sattler et al. J Cell Sci 2018;131:jcs218040
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