Filipin 3

Filipin staining is widely used as a method to measure unesterified cholesterol levels (31 (link)). Firstly, cells were seeded in coverslips and were cultured for 12–18 h. After washed by phosphate-buffered saline (PBS) three times, the cells were fixed via 4% paraformaldehyde for 20 min at room temperature. Afterwards, cells were treated with filipin III (0.1 mg/ml, Absin, China) for 30 min at room temperature. Besides, cells also staining with propidium iodide (PI, 0.35 μg /ml) for 5 min. Finally, filipin and PI staining fluorescence intensity were measured using a confocal laser scanning microscope (Leica sp5, Germany) at 405 nm.

DHCR24 (#2033, Cell Signaling Technology), Tau5 (AHB0042, Thermo), Phospho-Tau (Thr181) (ab254409, Abcam), Phospho-Tau (Ser199) (#29,957, Cell Signaling Technology), Phospho-Tau (Ser396) (#9632, Cell Signaling Technology), Actin (#2118, Proteintech), cavin1 (#18,892–1-AP, Proteintech), Phospho-Tau (Ser262)(ab131354, Abcam), Phospho-Tau (Thr231)(#4137, ABclonal), Ras(27H5)(#3339, Cell Signaling Technology), p44/42 MAPK (ERK1/2) (137F5)(#4695S, Cell Signaling Technology), Phospho-p44/42 MAPK (ERK1/2)(Thr202/Tyr204)(#4730S, Cell Signaling Technology), MEK1/2 (D1A5)(#8727, Cell Signaling Technology), Phospho-MEK1/2 (Ser217/221)(#9154, Cell Signaling Technology), U0126 (#9903, Cell Signaling Technology), Propidium Iodide (abs9105, Absin), Methyl-β-cyclodextrin (abs42021762, Absin), Filipin III (abs42018484, Absin), Oil Red O (CAS:1320–06-5, O0625-25G, Sigma), HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L)(SA00001-2, Proteintech), HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L)(SA00001-1, Proteintech).