Fusion capt advance software

To investigate the effects of lomerizine pretreatment on LPS-induced NLRP3 or p-GSK3α/β levels in vitro, BV2 microglial cells were treated with lomerizine (10 µM) or vehicle (1% DMSO) for 30 min (NLRP3) or 5.5 h (p-GSK3α/β) followed by LPS (200 ng/ml) or PBS for 30 min (p-GSK3α/β) or 5.5 h (NLRP3). BV2 microglial cells were harvested in lysis buffer (50 mM Tris, pH 7.4, 1% Triton X-100, 2 mM CaCl₂ and 2 mM MgCl₂) containing protease and phosphatase inhibitors and centrifuged at 12000 rpm for 15 min. Protein assay reagents (Bio-Rad Laboratories, Hercules, CA, USA) were used to quantify the protein concentration in the supernatant. For western blot analysis, 20 µg of protein sample was boiled at 100°C for 5 min, loaded onto an 8% SDS-PAGE gel, and separated by electrophoresis. The proteins in the gel were electrotransferred to a PVDF membrane (Millipore, Bedford, MA, USA), which was blocked in 5% skim milk and incubated with the primary antibody at 4°C overnight. The next day, the membrane was incubated with HRP-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rabbit IgG (1:10000, Enzo Life Sciences, Farmingdale, NY, USA), and proteins were detected by adding ECL Western Blotting Detection solution (GE Healthcare, Chicago, IL, USA). Images were acquired and analyzed by Fusion Capt Advance software (Vilber Lourmat).

For western blotting, BV2 microglial cells, primary astrocytes, or mouse brain tissues were lysed in ProPrep lysis buffer (iNtRON Biotechnology, Inc., Seongnam, Korea) and centrifuged at 12,000 rpm for 15 min. The protein concentration in the supernatant was quantified by reference to a standard solution of BSA. Next, 10 μg of protein was loaded onto an 8% SDS gel, separated by electrophoresis, and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% skim milk for 1 h at room temperature and then incubated overnight at 4°C with anti-p-AKT^ser473 (1:1000, Cell Signaling), anti-AKT (1:1000, Cell Signaling), anti-p-FAK (1:1000, Cell Signaling), anti-FAK (1:1000, Cell Signaling), anti-NF-kB (1:1000, Cell Signaling), or anti-PCNA (1:1000, Santa Cruz). Finally, the membrane was incubated for 1 h with HRP-conjugated goat anti-mouse IgG or HRP-conjugated goat anti-rabbit IgG (both 1:1000, Enzo Life Sciences, Farmingdale, NY, USA), and ECL Western Blotting Detection Reagent was added for detection (GE Healthcare, Chicago, IL, USA). Fusion Capt Advance software (Vilber Lourmat) was employed to acquire and analyze images.

The effects of tranylcypromine on LPS-induced ERK signaling were assessed in BV2 microglial cells treated successively with LPS (1 μg/mL) or PBS for 45 min and tranylcypromine (5 μM) or vehicle (1% DMSO) for 45 min. The cells were then prepared for western blotting by lysis in Cell Lysis Buffer (ProPrep, iNtRON Biotechnology, Inc., Seongnam, Korea). After centrifuging the lysate at 12,000 rpm for 15 min, the supernatant was collected. The protein samples were separated by SDS gel electrophoresis and then, electrotransferred to a polyvinylidene difluoride (PVDF) membrane, which was blocked with 5% skim milk or 5% BSA and incubated with anti-ERK (1:1000, Santa Cruz Biotechnology) or anti-p-ERK (1:1000, Cell Signaling) antibodies overnight. After incubating the membranes with horseradish peroxidase-conjugated secondary antibody for 1 h, detection was achieved with ECL Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA). Fusion Capt Advance software (Vilber Lourmat) was used to acquire and analyze images.

MDBK cells were seeded in 24-well plates and incubated to 80% confluence. The cells were infected with the EV-F7 SWUN-AB001 strain (MOI, 0.01) and incubated for a further 12 h or 24 h. Western blot analysis was then performed as previously described [11 (link)], using polyclonal antibodies against JNK/SAPK, phospho-JNK/SAPK, p38 MAPK, phospho-p38 MAPK, or GADPH (Cell Signaling Technology). Horse radish peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG antibodies were obtained from Abbkine. Densitometry values for the immunoblot signals were obtained from three separate experiments using FusionCapt Advance software (Vilber Lourmat).

Preparation of total protein was perfomed using the Qproteome™ Mammalian Protein Prep Kit (Qiagen) according to the manufacturer’s protocols. Determination of protein concentration was performed using the DC Protein Assay (Bio-Rad) according to the manufacturer’s protocols. 15 μg of total protein extracts per lane were loaded onto 8% polyacrylamide gels. Proteins were transferred onto Amersham™ Hybond™ ECL Nitrocellulose Membrane (GE Healthcare) by Semi-Dry blot. The membrane was blocked in 5% (m/v) nonfat dried milk powder in Tris buffered saline with Tween (TBST). Then the primary antibodies diluted in 5% (m/v) nonfat dried milk powder in TBST (mouse anti-human EGFR clone H11 (Dako), mouse anti-β-Actin clone AC-15 (Sigma-Aldrich)) or 5% BSA in TBST (polyclonal rabbit anti-HER2/ErbB2 (Cell Signaling)) were incubated. After HRP conjugated secondary antibody (Dianova) incubation, the membrane was incubated with ECL reagents (Thermo Scientific). Chemiluminescence detection and imaging was done with the Fusion Fx7 System and the FusionCapt Advance Software (Vilber Lourmat).

siRNAs against BCA3 (C11orf17) were purchased from Ambion (Thermo Fisher Scientific, s.r.o., Prague, Czech Republic). One day before transfection, HEK 293 cells were seeded in 12-well plate at the density 3 × 10⁵ cells/mL. Next day the cells were transfected with siRNA at various concentrations using siPORT (Ambion), according to the manufacturer’s instructions. At 24 h post the first transfection, the cells were transfected with HA-BCA3 expression vector (0.4 µg per well) using Fugene. At 24 h post-second-transfection, the cells were harvested and analyzed for presence of HA-BCA3 by Western blot analysis. In an experiment when the effect of BCA3 knockdown on HIV-1 release was analyzed, HEK 293 cells were grown in 60 mm plates and the transfection procedure was carried out as described above, with the exception that the psPAX2 vector was cotransfected with that encoding HA-BCA3. At 24 h post-second-transfection, the culture media were filtered through 0.45 µm filter and ultracentrifuged through a 20% sucrose cushion at 40,000 rpm for 1 h in a SW41 rotor. Both the viral pellet and producing cells were analyzed by Western blot and quantification of virus release was determined as a ratio of protein bands’ intensity of virion-associated CA to cell-associated CA by using Fusion CAPT Advance software (Vilber Lourmat, Marne-la-Vallée, France).