Fig. 5

Cystatin F dimers physically interact with Aβ to inhibit the internalization of Aβ by monocytes. A Schematic of the binding assay. B Plates were coated with 4 ng/μL dissolved Aβ40, Aβ42, Aβ40-1, and Aβ42-1 for 16 h. Recombinant cystatin F dimers were added to the wells for 3 h. The primary antibody for cystatin F and the secondary antibody coupled with HRP were used to generate a colour reaction, and the absorbance was measured at 450 nm. C, D The GST pull-down assay and His pull-down assay were used to analyse the interaction between the cystatin F dimer and Aβ. E Schematic diagram depicting the BiFC assay for detecting the interaction between the cystatin F dimer and Aβ. F The BiFC assay was carried out as described in the Materials and Methods section. Then, 200 ng/mL cystatin F dimer-VN and 200 ng/mL Aβ-VC protein were incubated at 37 °C for 2 h, and the fluorescence intensity was assessed at 529 nm. G Prediction of the amino acid interactions between cystatin F dimer and Aβ. H-J The potential amino acids on cystatin F that interacted with Aβ were mutated to alanine. The interaction was determined by a GST pull-down assay. K–L THP-1 cells were pretreated with 250 ng/mL cystatin F dimer protein or cystatin F (RTSTK) mutant protein for 30 min, 1 μg/mL Aβ42-Alexa Fluor 647 or Aβ42 was added for an additional 30 min, and the cells were subjected to K FCM and L ELISA analysis. M, N THP-1 cells were pretreated with 250 ng/mL cystatin F dimer protein or cystatin F (RTSTK) mutant protein for 30 min, and 1 μg/ml Aβ42-Alexa Fluor 555 or Aβ42-Alexa Fluor 647 were added for an additional 30 min, and the cells were observed by M TIRFM and subjected to N FCM. The data are presented as the means ± SD and were analysed using Student’s t test and one-way ANOVA. ^**p < 0.01, ^***p < 0.001. Scale bar: 5 μm