(A) Functional validation of LR-PCA. LR-PCA demonstrates insulin-induced recruitment of Akt to rafts. N2A cells were transiently transfected with LR-GLuc1/HA and Akt-GLuc2, and treated with indicated concentrations of insulin for 1 h at 37°C before measurement of LR-PCA signals 48 h post-transfection in live cells; P = 0.0041; n = 5. (B) Insulin activates downstream phosphorylation of Akt and GSK3β in N2A cells. Cells were transiently transfected with Akt-GLuc2 and treated with indicated concentrations of insulin for 1 h prior to protein extraction (48 h post-transfection). Cell extracts were analyzed on Western blots with phospho-Akt(Ser473) and phospho-GSK3β(Ser9) antibodies, GAPDH antibody was used as a loading control. (C) Recruitment of APP and its proteolytic fragments to rafts as detected by LR-PCA. N2A cells were transiently transfected with indicated constructs; luminescence signal was measured 48 h post-transfection in live cells; P = 0.0034; n = 6. (D) Acute cholesterol depletion decreases the association of APP-GLuc2 with LR-GLuc1/HA reporter. N2A cells were transiently transfected with LR-GLuc1/HA and APP-GLuc2, treated with 5 or 10 mM of mβCD at 37°C for 30 min. LR-PCA signal was measured 48 h post-transfection in live cells; P = 0.0005; n = 6. (E) Acute cholesterol loading increases the association of APP-GLuc2 with LR-GLuc1/HA reporter. N2A cells were transiently transfected with LR-GLuc1/HA and APP-GLuc2, incubated in serum-free media for 9.5 h prior to exposure to 10 or 100 µM mβCD/cholesterol for 30 min. LR-PCA signal was measured 48 h post-transfection in live cells; P = 0.0029; n = 6. The luciferase activity is expressed as normalized luminescence units (NLU). Each dot represents individual NLU value, horizontal lines represent medians and IQR is shown in gray. Statistical significance was assessed using Kruskal–Wallis with Dunn’s post-hoc test. *P < 0.05, **P < 0.01 and ***P < 0.001.