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. 2013 Jan 9;190(4):1797–1806. doi: 10.4049/jimmunol.1202472

FIGURE 5.

FIGURE 5.

Effect of rHMGB1 protein on the formation of 97Q-GFP aggregates. (A–C) SHSY5Y cells were transiently transfected with 97Q-GFP plasmid and then treated with rHMGB1 at the indicated concentrations. Distribution of 97Q-GFP aggregates was observed (A), and the number of aggregate-containing cells was counted among 100 GFP-positive cells (B). (C) The average pixel density of 30 aggregates was measured after treatment with rHMGB1. (D) SHSY5Y cells were transiently transfected with 97Q-GFP plasmid followed by rHMGB1 protein treatment. Cell lysates were analyzed for aggregate formation using filter-trap assay. The filter membrane was immunoblotted with anti-GFP Ab to check the amount of aggregates (top panel). Whole-cell lysates for exogenous HMGB1 protein that penetrated the cell membrane was analyzed using anti-His Ab (bottom panel). *p < 0.05 versus the rHMGB1-untreated group (n = 3).