Fig. 3.

Characterization of BIN1–Tau interaction in primary neuron cultures (PNC). a Representative immunoblots from neuronal extracts obtained at DIV7, DIV14, and DIV21 (in duplicate) showing BIN1 and total and phosphorylated forms of Tau (Tau1 for non-phospho Ser195/Ser198/Ser199/Ser202; PHF1 for p-Ser396/Ser404; RZ3 and AT180 for p-Thr231). b, c Relative changes in BIN1 and Tau protein levels and in Tau phosphorylation during neuronal maturation. d Representative images of PNC showing PLA spots and Tau immunolabeling during neuronal maturation. e, f Change in total PLA volume and PLA density during neuronal maturation. N = 3 independent experiments. g Correlation between total PLA volume and total Tau volume in a representative experiment. Each dot represents a confocal image. h Representative images of PNC under- and overexpressing BIN1, showing PLA and Tau and BIN1 immunolabeling. shNT: non-targeting shRNA. i Boxed areas in (h) are 2.4× magnified. j, k Total BIN1 volume and PLA density in PNC under- and overexpressing BIN1, normalized with respective controls (shBIN1 with shNT and BIN1iso1 with Mock). N = 3 independent experiments. In box plots, red bars, black squares, and red plus signs indicate sample median and mean, and outliers, respectively. Wilcoxon rank-sum test; *p < 0.05; **p < 0.01; ***p < 0.001