Ripa buffer

Caspase-3 activity was assessed in H₂O₂ or 6-OHDA-treated H9C2 and SH-SY5Y cells, respectively. The cells were washed and lysed with RIPA buffer (ELPIS-Biotech, Korea). The protein concentration was quantified, using a bicinchoninic acid (BCA) assay (Thermo Scientific, MA, USA). The cell lysates were diluted and transferred to black 384-well plates, followed by the addition of an equal volume of Ac-DEVD-AFC (Enzo, Farmingdale, NY, USA)-containing reaction buffer. After incubation, fluorescence intensity was measured at excitation and emission wavelengths of 400 and 505 nm, respectively, using a VarioskanTM Flash Multimode Reader (Thermo Scientific, MA, USA).

We washed HEI-OC1 cells with ice-cold PBS and suspended them in 100 μL Radioimmunoprecipitation assay (RIPA) buffer (Elpis Biotech, Daejeon, Korea) containing Protease Inhibitor Cocktail Set 1 (Calbiochem, La Jolla, CA, USA). We transferred the suspension into a pre-cooled 1.5-mL tube, which we then incubated on ice for 15 min, centrifuged at 12,660 g for 30 min at 4 °C, and vortexed for 1 min. We aspirated the resulting supernatant and placed it in a fresh tube on ice, discarding the pellet.

Whole cells were lysed in radio-immunoprecipitation assay (RIPA) buffer (ELPIS biotech, Daejeon, Korea) in the presence of a protease inhibitor cocktail. Extracted proteins were resolved by 10% or 12% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane blocked with 5% skim milk (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h. The membrane was then incubated with primary antibodies (1:1000) overnight at 4°C, washed with TBS-T, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:2000) for 2 h at room temperature. β-actin was used as the loading control. The bound antibodies were detected using a chemiluminescent HRP substrate (ECL, Millipore, Billerica, MA, USA). The blots were quantified using the Alliance Mini 4M (UVITEC).

Total cellular proteins were extracted from bone marrow-derived macrophages (BMDMs) or dorsal skin using RIPA buffer (ELPIS Biotech, Deajeon, Republic of Korea) with phosphatase and protease inhibitors, and 1 μg of protein was quantified using the Bradford assay [64 (link)]. The proteins were separated using 12% SDS-PAGE and moved to PVDF membranes. After blocking with 5% skimmed milk powder in 1X TBS containing 0.1% Tween-20 for 2 h, the PVDF membranes were incubated overnight at 4 °C with rabbit primary antibodies against NLRP3 (1:2000, AdipoGen Life Sciences, San Diego, CA, USA), mature IL-1β (IL-1β p17; 1:1000, Cell Signaling Technology), pro-IL-1β (IL-1β p31; 1:1000, Abcam, Cambridge, UK), cleaved caspase-1 (caspase-1 p20; 1:1000, AdipoGen Life Sciences), pro-caspase-1 (caspase-1 p48; 1:1000, Abcam), and β-actin (1:10,000, Bethyl Laboratories, Montgomery, TX, USA) [63 (link)]. The membranes were washed and then reacted with HRP-conjugated secondary antibodies at room temperature (20 ± 5 °C) for 2 h. The membranes were detected using enhanced chemiluminescence, and the target protein bands were observed manually by developing X-ray films or using the LAS-500 mini imager (General Electric, Boston, MA, USA). The expression levels of target proteins were analyzed using ImageJ (1.51j8).

HepG2 cells were cultured as described in Cell Culture and Treatment. Cells were treated with SCE or SA, and the plate was incubated for another 18 h. After incubation, both cell pellets and media were collected. Pellets were rinsed with DPBS and lysed with RIPA buffer (Elpis Biotech, Inc., Republic of Korea) containing protease and phosphatase inhibitor (Roche, Switzerland) in ice, before vortexing, centrifuging (15,000 ×g, 30 min, 4°C), and collection of the supernatant as a whole cell lysate. ELISA kits were purchased from Cell Biolabs Inc., (USA) and analysis of PCSK9 (STA 385) and LDLR (STA 386) was performed per the manufacturer’s instructions. Quantification of the protein in samples was determined in triplicate by BCA protein analysis (Pierce BCA Protein Assay Kit, Thermo Scientific, USA). Protein analysis was also performed according to the manufacturer’s instructions. PCSK9 and LDLR levels were normalized to total protein content.

Whole cells were lysed in RIPA buffer (ELPIS biotech, Daejeon, Korea) with a protease inhibitor cocktail. Extracted proteins were resolved by 12% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. Then proteins were blocked with TBS-T containing 5% skim milk (Santa Cruz Biotechnology) at room temperature for 1 h and incubated overnight at 4 °C with the primary antibody (1:1000). The PVDF membrane was washed in TBS-T and incubated for 2 h at room temperature with HRP-conjugated secondary antibody (1:2000). β-actin was used loading control for protein. The bound antibodies were detected using chemiluminescent HRP substrate (ECL, Millipore, Billerica, MA). The blots were exposed to Fuji medical X-Ray film RX-N (Fuji, Japan) or quantified using an Alliance Mini 4M (UVITEC Cambridge, UK).