Ab134113

For confocal microscopic co-localization studies, the cells were seeded onto BD Falcon Cultures Slides (Becton Dickinson). Next day the fibroblasts were washed, fixed with ice-cold methanol, blocked with 5% FBS in PBS and co-labelled rabbit monoclonal anti-NPC1 (1:100, ab134113, Abcam) and mouse monoclonal anti-LAMP2 (1:500, H4B4, Iowa Hybridoma Bank) antibodies at 4 °C overnight. Secondary antibodies were donkey anti-IgG anti-mouse alexafluor555 or anti-rabbit alexafluor488 conjugates (Pierce) diluted 1:1000. Leica SP8X laser scanning confocal system equipped with 470 nm–670 nm 80 MHz pulse continuum White Light Laser 2 and HC PL APO 63x/1.40 OIL CS2 (W.D. 0.14 mm) objective was used to image the samples. Image acquisition conditions were: excitation at 496 nm or 553 nm, one voxel 42.2 × 42.2 × 130 nm, 7 Z-steps (fulfilling Nyquist sampling theorem), Hybrid detectors at 503–553 nm or 566–650 nm. Acquired confocal images were deconvolved using theoretical point spread function in Huygens Professional software (Scientific Volume Imaging - SVI, Hilversum, The Netherlands). Overlay colocalization maps and Object Pearson’s coefficients were computed using Huscript (SVI), the grayscale maps were converted to colour coding look-up table (LUT) in Fiji/ImageJ software (NIH, Bethesda).

Tissues were homogenized (Ultra-Turrax TP, IKA Labortechnik, Wasserburg, Germany) on ice in 300 µl of RIPA lysis buffer (Thermo) per 100 mg of tissue with 1× protease inhibitor cocktail (Thermo) and incubated for 30 min. Lysates were centrifuged at 14 000g, 4°C for 20 min and overall protein concentrations of the supernatant was determined by Pierce BCA Protein Assay (Life Technologies). Samples were incubated at 37°C for 30 min in 1× LDS sample buffer (Life Technologies) and 1× sample reducing agent (Life Technologies), after which 40 µg of protein were loaded per well in a NuPAGE Bis–Tris 4–12% polyacrylamide gel for protein separation via SDS-PAGE electrophoresis. Proteins were transferred to PDVF membrane at 400 mA for 1 h and membrane was blocked for 1 h at 4°C with 5% BSA in TBS + 3% Tween 20. Membranes were subsequently incubated overnight at 4°C with primary antibodies for NPC1 (1:10 000, ab134113, Abcam) and β-tubulin (1:2000, ab6161, Abcam) with 3% BSA in TBS + 3% Tween 20. After 3 washes in TBS, antibody staining was revealed using HRP-conjugated goat anti-rabbit IgG (1:2000, ab6721, Abcam) and goat anti-rat IgG (1:10 000, ab97057, Abcam) incubated for 2 h at RT in TBS + 3% Tween 20 with 3% BSA. Blots were developed with ECL system (SuperSignal West Pico, Life Technologies) and imaged using a Genegnome imager (Syngene, Cambridge, UK).

Cells grown to near confluence on 6 cm-dishes were lysed, separated by SDS gel electrophoresis and blotted onto polyvinylidene difluoride membranes. Antibodies directed against Abca1 (ab18180), LDL receptor (ab30532), HMG-CoA reductase (ab174830), SREBP-2 active fragment (ab30682), NPC1 (ab134113), and APP (ab32136) were obtained from Abcam (Cambridge, UK). Antibodies to AMPKα (#2603 S) and phospho-Thr172-AMPKα (#2535 S) were from Cell Signaling Technology (Danvers, MA, USA), while anti-β-actin (A5441) was from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany). HRP-conjugated secondary antibodies were from GE Healthcare (Freiburg, Germany). The enhanced chemiluminescence system was from Millipore Corporation (Billerica, MA, USA).