Hrp substrate

Cells were ruptured on ice using a RIPA lysis kit (ATTO Corporation), lysates were clarified by centrifugation, and samples were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred onto polyvinylidene (PVDF) membranes (Bio-Rad), which were then incubated with antibodies against NOX4 (1/1000 dilution; Abcam), β-actin (1/2000 dilution; Sigma-Aldrich), PI3Kδ (1/1000 dilution; Cell Signaling Technology), and GAPDH (1/3000 dilution; Cell Signaling Technology) overnight at 4 °C. After washing with PBS-T, membranes were stained with peroxidase-conjugated goat anti-rabbit IgG (Abcam) or peroxidase-conjugated rabbit anti-mouse IgG (Abcam). Target proteins were then detected using the chemiluminescent HRP Substrate (Thermo Fisher Scientific).

The membrane protein expression, turnover and recycling were measured using the PM epitope labelling and cs-ELISA in live cells for kinetic studies^{5 (link),12 (link),28 (link),59 (link)}. Internalization was measured for 5 min, recycling 20 min and stability for indicated times at 37 °C. Transferrin-HRP or HRP-conjugated secondary antibody (Ab) was measured either by luminescence using HRP-Substrate (SuperSignal West Pico, Thermo Fisher Scientific) or Ampilite (ATT Bioquest, CA, USA) or Amplex Red assay (Thermo Fisher Scientific).

To analyze the protein contents of engineered LPs, LP-containing supernatants were centrifuged through a 20% (wt/vol in PBS) sucrose cushion. LPs were lysed in the presence of a protease inhibitor. The viral proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to the PVDF membrane. Membranes were blocked by 5% fat-free milk dissolved in TBS/0.05% Tween-20 for 1 hr and incubated with an HA monoclonal antibody (Covance, Princeton, New Jersey) overnight at 4°C. The membranes were incubated with anti-mouse secondary antibodies (Dako, Glostrup, Denmark) and visualized by enhanced chemiluminescence (ECL) using a HRP substrate (Thermo Scientific). The HA monoclonal antibody was washed away by stripping buffer (Thermo Scientific), and the PVDF membrane was re-used for incubation with a HIV-1 p24 polyclonal antibody (Thermo Scientific) and later peroxidase-conjugated anti-rabbit secondary antibody (Dako).

Streptavidin-coated plates (Greiner) were loaded with biotinylated NI301A and blocked before the addition of ATTR or TTR samples in duplicates for 2 h incubation at room temperature. After washing, bound ATTR was detected with an HRP-conjugated NI301A antibody in combination with luminescent substrate (Biotinylation kit, HRP-labeling kit, and HRP substrate from ThermoFisher). Data were fitted using 4PL with 1/y² weighting.

Cell extracts were harvested by direct addition of 2X SDS sample buffer (1610747, Bio-Rad Laboratories, Alcobendas, Spain) containing 100 mM DTT to the culture plate and incubation on ice for 10 min, followed by collection in 1.5 mL eppendorf tubes and denaturation for 5 min at 95°C. Lysates were subjected to SDS-PAGE in Criterion TGX Stain-Free Precast Gels (567–8124, Bio-Rad Laboratories) and transferred to nitrocellulose membranes (IB301001, Thermo Fisher Scientific) by iBlot Dry Blotting System (Thermo Fisher Scientific). Membranes were blocked in 5% milk in TBS with 0.05% Tween-20 for 1 h at RT before addition of primary antibodies (SRRM4, HPA052783, Sigma-Aldrich Quimica S.A.; GAPDH, sc-47724, Santa Cruz Biotechnology [Dallas, Texas, USA]) diluted 1:1,000 in blocking buffer, followed by overnight incubation at 4°C. Membranes were washed 3 × 5 min in TBS-T, followed by 1 h incubation at RT in secondary antibodies (Anti-Rabbit: A0545; Anti-Mouse: A6782, Sigma-Aldrich Quimica S.A.) diluted 1:10,000 in blocking buffer, and 3 more 5 min washes with TBS-T. Finally, membranes were developed by the addition of HRP substrate (34096, Thermo Scientific, [Fisher Scientific, Madrid, Spain]) and exposed using a LAS-3000 Imaging System from Fujifilm (Tokyo, Japan).