Semi dry transfer unit

To determine the protein expression, RAW264.7 cells were seeded at a cell density of 5 × 10⁴ cells/plate in a 60π plate, and then subjected to pre-treatment for 1 h at 37 °C with and without LPS (1 μg/mL; Sigma-Aldrich) and PUG treatment with 4 and 6 μM for 24 h. After this, RIPA buffer (iNtRON Biotechnology, Incheon, Korea) was added to the cells to lyse and harvest the protein. Then the concentration of protein was estimated by the Pierce™ BCA protein assay kit (Thermo Scientific). To separate the proteins, an equal concentration of protein (10 μg) was loaded in SDS-PAGE. Then the gel was transferred to PVDF membranes via a Semi-Dry Transfer unit (Atto Corporation, Tokyo, Japan). The transferred membrane was then blocked for 1–2 h at room temperature with 5% Bovine Serum Albumin (BSA) and followed by incubation at 4 °C overnight with diluted (1:1000) primary antibodies. The membranes were washed with 1X TBST solution and probed with the horseradish peroxidase-conjugated secondary antibody (1:5000) anti-mouse (cell signaling; cat. No. A90-116P) for β-actin and anti-rabbit (cell signaling; cat. No. A120-101P) to the rest of the proteins at room temperature for 2 h and washed again. Finally, the blots were detected under detection system (Bio-Rad Laboratory), after developing the ECL solution. Protein densitometry was evaluated using the ImageJ software program.

Cells were planted at a cell density of 5 × 10⁴ cells/plate in a 60π plate to evaluate protein expression. Cells were treated for 1 h with 10, 20, and 40μM PUR for 24 h at 37 °C. Cells were subsequently collected and lysed with radioimmunoprecipitation assay (RIPA) buffer (iNtRON Biotechnology, Seongnam, Republic of Korea), which included [50 mM Tris-HCl (pH 8.0), 0.5% sodium deoxycholate, 1 mM EDTA, 150 mM NaCl, 0.1 SDS, and 1% NP40]. The protein concentration was then calculated using a Pierce^TM BCA protein assay kit (Thermo Scientific). SDS-PAGE was used to separate the proteins (10 g). The gel was then transferred to PVDF membranes using a semi-dry transfer unit (Atto Corporation, Tokyo). After blocking the membranes for 1–2 h at room temperature with 5% bovine serum albumin (BSA, Irving, TX, USA; Thermo Fisher Scientific), the membranes were incubated overnight at 4 °C with diluted (1:1000) primary antibodies. Then, the membranes were washed with 1X TBST solution before being incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (1:5000) for 2 h at room temperature. The blots were then washed with 1X TBST and developed using an electrochemiluminescence (ECL) detection system (Bio-Rad Laboratory, Hercules, CA, USA). ImageJ software (U.S. National Institutes of Health, Bethesda, MD, USA) was used to evaluate protein densitometry.