Sample reducing agent

Western blot analysis was performed in LOVO and SW480 cells using the following primary antibodies: GLUT1 (21829-1-AP, Proteintech) and α-tubulin (T 6074, Sigma-Aldrich). Briefly, the cell pellet was mixed with LDS sample buffer (Life Technologies) and sample reducing agent (Life Technologies), according to the manufacturer’s protocol. Proteins extracted were loaded onto a SDS-PAGE (Novex 4–12% Bis–Tris gel, Life Technologies), transferred to a nitrocellulose membrane by iBlot (Life Technologies), and blocked 1 h with 1× TBST buffer (Fisher Scientific) with 5% w/v nonfat dry milk. The membranes were incubated in blocking buffer at 4 °C overnight with primary antibody against GLUT1 (1:1000) or 1 h at room temperature for α-tubulin (1:3000). Subsequently, the membranes were washed three times with Tris-buffered saline with Tween-20 (TBST) and incubated 1 h with secondary antibodies (1:5000; anti-rabbit sc-2004, Santa Cruz Biotechnology; or 1:3000; anti-mouse A9044, Sigma-Aldrich). The signal was obtained using the Novex ECL Chemiluminescent Substrate Reagent kit (WP 20005, Invitrogen) and the images were developed and quantified using the Chemidoc System (Bio-Rad).

PASMCs were stimulated with rhOPG (50 ng ml⁻¹) (R&D systems), alongside quiesced cells (negative control) for 10 and 60 min, before lysing. Cell lysates were mixed with sample buffer (Life Technologies, Carlsbad, CA, USA) and sample reducing agent (Life Technologies), denatured by heating and subjected to gel electrophoresis. The membranes were then incubated with primary antibodies against phospho-CDK4, phospho-HSP27, total mTOR, phospho-mTOR (1:500) and GAPDH (1:1000) (Cell Signalling Technology), CDK5 (1:500) (Abcam), or β-actin (1:1000) (Santa Cruz Biotechnology, Heidelberg, Germany). Membranes were then incubated with anti-Rabbit IRDye 800CW and anti-Mouse IRDye 800CW (Li-COR, Lincoln, NE, USA) and signal detection and band density quantification was performed using the LiCOR Odyssey SA system.

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).