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. 2020 Feb 18;219(3):e201911126. doi: 10.1083/jcb.201911126

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© 2020 Itakura et al.

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Figure S4. — The conserved basic amino acids in Clusterin for secretion and internalization. (A) Some basic residues in Clusterin are required for secretion. HEK293 cells transiently transfected with the indicated CluRG vectors were cultured for 3 d, and conditioned medium and cell lysates were analyzed by immunoblotting. Clusterin mutants colored in blue showed reduced cleavage efficiency and secretion. Note that the expression of CluRG by the cytomegalovirus (CMV) promoter increased pre-Clusterin expression in the cell due to overexpression. (B) The indicated Clusterin-RG mutants were mixed with recombinant Luc in serum-free medium and incubated at 42°C for 20 min. HEK293 cells were cultured in the mixtures for 14 h at 37°C and analyzed by flow cytometry. The bar graph shows the relative RFP intensity in a cell normalized to that of Clusterin WT-RG (n = 3). The data are presented as the mean ± SEM. The combination of the R282Q, R286Q, R289Q, K123Q, R127Q, R130Q, and R138Q mutations (BQs) most significantly reduced Clusterin internalization.