Figure 3.

apoAlt a 1 exerts proteolytic function and decreases the labile iron pool upon clathrin-mediated uptake. (a) Recombinant Alt a 1 (10 μg) in the absence (apo, red box) or presence of iron–quercetin (holo, blue box) and with or without Zn/Ca were left uncooked or cooked before proteins were separated on 4–20% SDS-PAGE under non-reducing conditions and proteins bands were visualized by silver stain. (b) Representative inverted image of casein zymogram with summary of quantification of 2 independent experiments. (c) Representative inverted image of gelatin zymogram with summary of quantification of 2 independent experiments. (d) Summary of mean fluorescence intensity MFI of the calcein signal in THP1 cells incubated 18 h with apo− and holoAlt a 1 from three independent experiments (e) AhR activation was measured after 18 h in AZ-AhR cells treated with 10 μM of iron–quercetin complexes alone or in combination with 0.5, 1, and 1.5 μM Alt a 1. Experiments were performed at least 3 times independently and normalized to medium alone. Summary of 2 h-starved THP1 cells incubated for 1 h at 4 °C or 38 °C with fluorescent-labeled apo/holoAlt a 1 and determined by flow cytometry (f) % binding (g) APC-mean fluorescence intensity of Thp1-cells, (h) incubated for 1 h with labeled apoAlt a 1 in the presence of Pitstop 2 or Dyngo4 and (i) for 1 h with labeled holoAlt a 1 in the presence of Pitstop 2 or Dyngo4. Statistical analyses of zymograms in (b,c) from two independent experiments were analyzed by RM one-way-ANOVA followed by Tukey’s multiple comparison test with a single pooled variance, (d,h,i) was analyzed by Mixed-effect analysis with the Geisser–Greenhouse correction, followed by Tukey’s multiple comparison test, (e–g) was analyzed with RM two-way Anova with the Geisser–Greenhouse correction and the Tukey’s multiple comparison tests with a single pooled variance. * p < 0.05, ** p < 0.01.