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. 2016 Mar 1;291(18):9554–9565. doi: 10.1074/jbc.M115.700518

FIGURE 2.

FIGURE 2.

Serum albumin stimulates microneme secretion and is enhanced by ethanol. A, MIC2-GLuc-C-myc secretion assay. Purified RH-MIC2-GLuc-C-myc parasites were stimulated with serial dilutions of ethanol alone, serum alone, or ethanol in the presence of 1% serum for 10 min at 37 °C. Release of MIC2-GLuc-C-myc in ESA was determined by luciferase assay. The graph indicates the average and S.D. of triplicate wells for each treatment dilution and is representative of two independent experiments with similar outcomes. B, MIC2-GLuc-C-myc secretion assay comparing whole serum to filtered serum. Purified RH-MIC2-GLuc-C-myc parasites were incubated for 10 min at 37 °C with buffer alone (mock), 1% serum (whole), 30-kDa MWCO filtered 1% serum (<30 kDa), 30-kDa MWCO filtered 1% serum + 0.18% bovine serum albumin (BSA) add back (<30 kDa + BSA), or 0.18% serum albumin alone (BSA). Release of MIC2-GLuc-C-myc in ESA was determined by luciferase assay. The graph indicates the average and S.D. of triplicate wells for each treatment and is representative of two independent experiments with similar outcomes. *, p ≤ 0.05 versus mock by one-way analysis of variance with Tukey's multiple comparison test. C, MIC2-GLuc-C-myc secretion assay. Purified RH-MIC2-GLuc-C-myc parasites were stimulated with serial dilutions of serum or equivalent amounts of serum albumin alone for 10 min at 37 °C. Release of MIC2-GLuc-C-myc in ESA was determined by luciferase assay. The graph indicates the average and S.D. of triplicate wells for each treatment dilution and is representative of two independent experiments with similar outcomes. D, MIC10-GLuc-C-myc secretion assay. Purified RH-MIC10-Gluc parasites were treated with or without 1% BSA for 10 min at 37 °C. ESA was collected and subjected to a luciferase assay. *, p ≤ 0.05, unpaired Student's t test. E, MIC2-GLuc-C-myc secretion assay. Purified RH-MIC2-GLuc-C-myc parasites were stimulated with serial dilutions of ethanol with or without 1% serum albumin for 10 min at 37 °C. Release of MIC2-GLuc-C-myc in ESA was determined by luciferase assay. The graph indicates the average and S.D. of triplicate wells for each treatment dilution and is representative of two independent experiments with similar outcomes. F, MIC2-GLuc-C-myc secretion assay comparing IC versus EC buffer in the presence or absence of BAPTA-AM. Purified RH-MIC2-GLuc-C-myc parasites resuspended in IC buffer or EC buffer were pretreated with BAPTA-AM or vehicle control and incubated for 10 min at 37 °C with buffer alone (mock), 1% BSA, 1% ethanol, or 1% BSA + 1% ethanol. Release of MIC2-GLuc-C-myc in ESA was determined by luciferase assay. The graph indicates the average and S.D. of two independent experiments consisting of triplicate wells for each treatment. *, p ≤ 0.05 versus mock by two-way analysis of variance with Tukey's multiple comparison test. All BAPTA-AM treatments were significantly lower compared with their corresponding dimethyl sulfoxide (DMSO) controls.