Ecl reagent

FPLC HDL fractions (25 µL), plasma (1 µL), or liver extract (25–50 µg) were reduced with β-mercaptoethanol/Laemmli buffer, separated on 4–20% mini Protean TGX gels (Bio-Rad), transferred to a PVDF membrane, and probed with primary antibodies for mouse apoAI (Abcam-ab20453), mouse apoD (Santa Cruz-sc34763), SR-BI (Novus Biologicals-NB400-104), LDLR (BioVision-3839-100), and β-actin (Thermo Scientific-MA-91399). Proteins were detected with ECL reagent (Promega) and the pixel intensities of bands were quantified by the Image J program (NIH). For total protein analysis, 10–20 µg of total protein was loaded on 4–20% mini TGX gel and gel stained with SimplyBlue SafeStain (Invitrogen) to detect apolipoproteins' levels in pooled FPLC fractions.

Polyclonal rabbit antisera against the unique C-terminal domains of the NiV P, W and V proteins and to the NiV C protein were produced by GenScript (Piscataway, NJ). Overall, 1.2 × 10⁶ Vero cells per well were seeded in a six-well plate and infected with rNiV_M-wt, rNiV_M-W^ko or rNiV_M-V^ko at an MOI of 0.01. The cells were harvested at 40 h.p.i. in 1 ml of 2 × Laemmli sample buffer (Bio-Rad, Hercules, CA) and heated to 95 °C for 20 min. Samples were then run on a denaturing 4–12% SDS–PAGE gel (Bio-Rad). Proteins were transferred from the gel on polyvinilidene fluoride (PVDF) membranes and blocked in TTBS (100 mM Tris-HCl pH 7.5, 0.9% NaCl, 0.1% Tween 20) with 5% skim milk. PVDF membranes were incubated with polyclonal rabbit antisera against P, V, W and C described above, diluted in TTBS with 5% milk (P: 1:10,000; V: 1:5,000; W: 1:5,000; C: 1:500) for 1 h at room temperature and washed three times in TTBS. The membranes were then incubated with anti-rabbit IgG conjugated to horseradish peroxidase (Sigma-Aldrich; 1:20,000 dilution) for 1 h at room temperature, washed three times in TTBS, incubated with ECL reagent (Promega) for 5 min and imaged with a VersaDoc (Bio-Rad).

Total protein extracts were prepared by boiling the cells in SDS sample buffer for 10 min at 95 °C. The proteins were then separated by 10% SDS-PAGE and transferred onto Immobilon-P^SQ membranes (Millipore). Western blotting was performed by incubating the membranes overnight at 4 °C with primary antibodies against the following proteins: Brm (ab15597; Abcam), BRG1 (sc-10768; Santa Cruz), Halo tag (G928A; Promega), HA tag (#3724; Cell Signaling), and β-actin (sc-47778; Santa Cruz). After three washes with Tris-buffered saline containing Tween 20, the membranes were incubated with secondary antibodies [donkey anti-rabbit-horseradish peroxidase (AP182P) or donkey anti-mouse-horseradish peroxidase (AP192P); Millipore] for 1 h at room temperature. Signals were detected using ECL reagent (Promega) or ImmunoStar LD (Wako). Amounts of charged protein samples were roughly normalized to β-actin.

For analysis of HGF-dependent MET activation, sub-confluent cell monolayers were maintained in the absence of serum for 24 h, then stimulated with HGF (100 ng/mL—R&D Systems) for 15 min at 37 °C. Cell lysates were obtained using Laemmli buffer. Equal amounts of total proteins (20 µg/sample) were resolved by SDS-PAGE and transferred to 0.2 µm nitrocellulose Trans-Blot Turbo TM membranes (Thermo Fisher Scientific Inc.). For protein detection, the following antibodies were used: anti-MET phospho-Tyr1234/1235 (D26); anti-MET (D1C2); anti-pAKT (Ser473), anti-AKT, anti-pERK1/2 (Thr202/Tyr204), anti-ERK1/2 polyclonal Abs (all from Cell Signaling Technology, Beverly, MA, USA); anti-vinculin (clone hVIN-1) and anti-alpha actin (both from Sigma Life Science, St. Luois, MO, USA). All antibodies were applied according to the protocols supplied by the manufacturers. After incubation with appropriate HRP-conjugated secondary antibodies (all from GE Healthcare) and the ECL reagent (Promega Corp.), Western blot bands were detected by ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Segrate, Italy).

Total protein extracts were prepared by boiling the cells in SDS sample buffer for 5 min at 95°C. The proteins were then separated by 10% SDS-PAGE and transferred onto Immobilon-P PVDF membranes (Millipore). Immunoblotting was performed by incubating the membrane overnight at 4°C with primary antibodies against the following proteins: Brm (ab15597; Abcam), BRG1 (sc-10768; Santa Cruz), EGR1 (#4153; Cell Signaling), CD44 (#3570; Cell Signaling), MET (#8198; Cell Signaling), CAV1(#3267; Cell Signaling), CAV2 (#8522; Cell Signaling), SIRT1 (sc-74465; Santa Cruz), GSK3β (#12456; Cell Signaling), RELA (ab7971; Abcam), PTEN (#9552; Cell Signaling), IKKβ (#8943; Cell Signaling), and β-actin (sc-47778; Santa Cruz). After three washes with Tris-buffered saline (TBS) containing Tween 20, the membranes were incubated with secondary antibodies [donkey anti-rabbit-horseradish peroxidase (AP182P) and donkey anti-mouse-horseradish peroxidase (AP192P)]; Millipore) for 1 h at room temperature. Signals were detected using ECL reagent (Promega) or ImmunoStar LD (Wako). Amounts of charged protein samples were roughly normalized to β-actin. Relative protein amounts were quantified by Ez-Capture MG (ATTO) using Multi Gauge V3.2 (Fuji Film) software after strict normalization by the internal control, β-actin.