Odyssey infrared fluorescent system

After 72 hours of culture, total cell extracts were lysed in Laemmli Buffer. Protein samples were resolved by SDS‐PAGE and immunoblotted with antibodies: anti‐PPARG (sc‐7196, Santa Cruz), anti‐GFP (AB0020, Sicgen), anti‐vinculin (V9131, Sigma‐Aldrich) and/or anti‐actin (A5441, Sigma‐Aldrich). After 1 hour incubation with the appropriate Alexa Fluor‐conjugated secondary antibody (680 or 800 conjugate, Molecular Probes), blots were revealed using the Odyssey infrared fluorescent system (Li‐Cor). Signal intensity was quantified using Image J. The arbitrary pixel densities of each protein were normalized to actin.

Total proteins were isolated from cells incubated with RTV, LPV or DMSO using Lysis Buffer (NP40 Cell Lysis Buffer; Invitrogen^TM, Carlsbad, CA, USA) combined with a protease inhibitor cocktail 100× (1× final; Protease Inhibitor Cocktail Set I, Calbiochem^®, EMD Chemicals Inc., Merck KGaA, Darmstadt, Germany) and a phosphatase inhibitor cocktail 50× (1× final; Phosphatase Inhibitor Cocktail 50× Set V, Calbiochem^®, EMD Chemicals Inc., Merck KGaA, Darmstadt, Germany). Proteins (20 µg) were loaded on 4–15% gradient gel (Mini-PROTEAN^® TGX^TM Precast Gels, BIORAD^®, Hercules, CA, USA), and were then transferred on nitrocellulose membrane which was blocked with TBS 1×-Milk 5%-Tween 0.1% solution. The resulting proteins blots were probed with anti-MLN64, anti-P450SCC, anti-HSD3B1, anti-OPA1, anti-Mfn2, anti-GRP78, monoclonal mouse anti-actine or anti-vinculine antibodies (references and concentration in Table 1) [11 (link),17 (link)]. Actine or vinculine were used as loading control. Addition of secondary goat anti-mouse antibody conjugated with DyLight 680 (1/15,000, #35518 Thermo Fisher Scientific, Waltham, MA, USA) or secondary goat anti-rabbit antibody conjugated with DyLight800 4× PEG (1/15,000, SA5-35571 Thermo Fischer Scientific, Waltham, MA, USA) allowed blots revelation using Odyssey infrared fluorescent system (LI-COR).

Total cell extracts were prepared as previously described by41 (link). Plasma membrane protein fractions were obtained using the ProteoExtract Native Membrane protein Extraction Kit (Merck Millipore, France) accordingly do the manufacturer’s protocol. Protein samples were resolved by SDS-PAGE and immunoblotted with antibodies to AnxA1, AnxA2, AnxA6 (0.2 μg/ml each, Santa-Cruz Biotechnology, Germany), AnxA5 (2 μg/ml, Sigma-Aldrich, France), α-Catenin (0.5 μg/ml, Life Technologies, France), β-Catenin (0.25 μg/ml, Life Technologies, France), E-Cadherin (0.25 μg/ml, Life Technologies, France), ezrin (0.5 μg/ml, Life Technologies, France), actin (0.8 μg/ml, Sigma-Aldrich, France), or GFP (1 μg/ml, Clontech, France). After incubation with appropriate DyLight Fluor-conjugated secondary antibody (680 or 800 conjugate, Life Technologies, France) blots were revealed by using Odyssey infrared fluorescent system (Li-Cor, France). Conversely, blot with cell membrane factions were incubated with reversible protein stain (Thermo scientific, France).

Cortical or hippocampal tissue was weighed, homogenized and incubated for 30 min in RIPA buffer containing protease inhibitors. The homogenate was then centrifuged at 13,000 g for 10 min at 4 °C. Protein samples were separated on premade 4 – 12% SDS-PAGE gels and were transferred to PVDF membranes (Bio-Rad). After incubation with primary antibodies, including anti-Aβ₄₂ (1:1,000, Invitrogen, USA), anti-BACE1 (1:200, Bioss, China), anti-APP (1:200, Bioss, China), anti-COX-2 (1:200, Cayman, USA), and anti-MAGL (1:200, Cayman, USA), overnight at 4 °C, the membranes were blotted with IR Dye 800 CW-conjugated secondary antibody (1:5000, LiCor Biosciences, USA) at room temperature for 1 h. The densitometry of the bands was scanned and measured using a LI-COR Odyssey Infrared Fluorescent System. The density of each band was quantified using Image-pro Express software, version 6.0 (Media Cybernetics, USA) and was normalized to the corresponding β-actin (1:1000, Cell Signaling, USA) value to account for variations in loading.

BEAS-2B cell were lysed in RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) containing PMSF (1 mM). After centrifugation at 20000 x g for 10 min, the supernatant was collected and its concentration was determined using an Enhanced BCA Protein Assay Kit (P0009, Beyotime, Shanghai, China). Forty micrograms of proteins were separated by SDS-PAGE using 8% gels for detecting TRPM2 and LAMP1, or 12% gels for SQSTM1, LC3B and Cathepsin-D. β-actin was used as the protein loading control. Proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilon-P, Millipore, MA, USA), and blocked in 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (v/v) (TBST) for 2 h at room temperature. The membranes were incubated with primary antibodies for TRPM2 (ab96785, 1:1000 dilution, Abcam, USA), LAMP1 (#9091, 1:1000 dilution, CST, USA), SQSTM1 (PM045, 1:1000 dilution, MBL, Japan), LC3B (#12741, 1:1000 dilution, CST, USA), Cathepsin-D (ab75852, 1:1000 dilution, Abcam, USA), and β-actin (A5316, 1:5000 dilution, Sigma, USA) overnight at 4 °C. After subjected to three 5-min washes in TBST, the membranes were incubated with secondary antibodies conjugated with various fluorophores (926–68,020, anti-mouse; 926–32,211, anti-rabbit; 1:5000 dilution, LI-COR, USA), and proteins were visualized and analyzed using a LI-COR Odyssey Infrared Fluorescent System.

Proteins were extracted in ice-cold lysis buffer (1% Nonidet P-40, 1 mM EDTA, 125 mM sodium fluoride, 0.5 mM sodium vanadate, 2.5 μg/mL of aprotinin, 5 μg/mL of pepstatin, 50 μg/mL of leupeptin, 25 μM PMSF, and 25 μg/mL of trypsin inhibitor). The protein concentration was determined according to the Bradford method using BSA as the standard protein [67 (link)]. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis was performed on 50 μg protein samples using 12% resolving/4% stacking Tris-HCl gels. Electrophoresis proteins were transferred to nitrocellulose membranes using Bio-Rad Mini Trans-Blot Electrophoretic Transfer Cell Instruments. Membranes were blocked in 3% BSA solution for 1 h at room temperature and incubated overnight at 4 °C with antibodies to targeted proteins (anti-GAPDH antibody, CST; anti-Col1a1 antibody, Bioss; anti-Col3a1 antibody, Bioss; anti-TGFβ1 antibody, Proteintech; anti- NADPH oxidase 4 (NOX-4)antibody, BBI; anti-Smad 3 antibody, BBI; and anti-P-Smad 3 antibody, BBI). After washing, the membranes were incubated with fluorescently labeled secondary antibody (1,5000) (IRDye 800CW goat anti-rabbit IgG (H + L), LI-COR), scanned, and detected with the LI-COR Odyssey Infrared Fluorescent System.