Ecl solution

Protein samples from both the mouse cerebrum, U373-MG, and SH-SY5Y cells were extracted using a protein lysis buffer (T-PER reagent, 78510, Thermo Fisher Scientific Inc., Waltham, MA, USA). The protein concentration was determined using the Bradford assay with PRO-Measure solution (#21011, iNtRON Biotechnology, Kirkland, WA, USA). For electrophoresis, SDS-PAGE was performed on 10% or 12% polyacrylamide gels, depending on the protein size. The separated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane. To prevent non-specific binding, the membranes were blocked for 1 h using 3% Bovine Serum Albumin (BSA100, 9048-46-8, LPS solution, Daejeon, Republic of Korea) in diluted TBS-T buffer (04870517TBST4021, LPS solution). Primary antibodies were applied overnight at 4 °C, followed by washing the membranes with TBS-T. Subsequently, secondary antibodies were applied using the same procedure. The results were visualized using ECL solution (XLS025-0000, Cyanagen, Bologna, Italy), and Chemi Doc (Fusion Solo, VilberLourmat, Collégien, France) was used for imaging. All primary antibodies were diluted at a ratio of 1:2500 in 5% w/v BSA, while the secondary antibodies were diluted at the same ratio in 5% w/v skim milk. Detailed information on the primary and secondary antibodies is provided in Table 1.

Total liver and hep3B cell samples were homogenized by protein lysis buffer [T-PER (#78510, Thermo)] containing proteinase inhibitor (PMSF). Homogenized samples were quantified by Bradford assay with PRO-Measure solution (Intron, #21011) and proceeded to protein electrophoresis (SDS-PAGE) after 5 min of boiling in 100 °C. Gels were blotted by wet transfer with Bio-Rad Power Pac in 350 mA. Membranes were blocked for 1 hour in Skim milk and incubated with a primary antibody (see above) for overnight, 4 °C. After 3 times of washing with PBS-T or TBS-T, membranes were incubated with secondary antibodies (AE-1475 goat anti-rabbit, BS-0296G-HRP goat anti-mouse, Bioss) for 4 hours diluted with 1:5000 in skim milk or BSA. Results were detected with ECL solution (XLS025-0000, Cyanagen) and Chemi Doc (Fusion Solo, Vilber Lourmat).

Liver and Hep3B cells were lysed and sonicated for protein extraction. Protein samples were subjected to 8–12% SDS-PAGE after 5 min of boiling at 100 °C. Gels were blotted by wet transfer with Bio-Rad Power Pac at 350 V for 1 to 2 h. PVDF membranes were blocked for 1 h in bovine serum albumin (BSA) and incubated with primary antibodies overnight at 4 °C. Membranes were then incubated with secondary antibodies diluted with 1:10,000 in BSA overnight at 4 °C. The results were detected with enhanced chemiluminescence (ECL) solution (Cyanagen) and ChemiDoc (Fusion Solo, Vilber Lourmat). Primary polyclonal antibodies used were: rabbit anti-β-actin (sc-130656, Santa Cruz Biotechnology, USA) and rabbit anti-PEPCK (10004943, Cayman Chemical, USA). Rabbit monoclonal antibody to PGRMC1 (#13856) was purchased from Cell Signaling Technology, Inc. Rabbit monoclonal antibody to phosphor CREB (ab32096, Abcam), CREB (ab32515, Abcam) were used. The secondary antibody used was mouse anti-rabbit IgG (211-032-171 anti-rabbit, Jackson laboratory).

Protein samples, including LV tissues of the hearts and H9c2 cells, were extracted using T-PER reagent (78510, Thermo Fisher Scientific, Waltham, MA, USA), which is a protein lysis buffer. Through a Bradford assay, the sample concentration was quantified using PRO-Measure solution (#21011, Intron, Seoul, Korea). SDS-PAGE electrophoresis was performed on 10% polyacrylamide gels and the gels were transferred to a PVDF membrane. Following the transfer, the membranes were blocked using 3% BSA (9048-46-8, LPS Solution, Chisinau, Moldova) with diluted TBS-T buffer (04870517TBST4021, LPS solution). Primary antibodies (Table 1) dissolved with 3% BSA were operated overnight in 4 °C. The membranes were washed three times with TBS-T and secondary antibodies (Table 1) were operated in identical fashion to primary antibodies. The blotting results were detected with ECL solution (XLS025-0000, Cyanagen, Bologna, Italy) and Chemi Doc (Fusion Solo, Vilber Lourmat, Marne-la-Vallée, France).

Homogenization of foot ulcer samples using POLYTRON-800 homogenizer was done, using RIPA buffer (Radio Immuno Precipitation Assay) for lysis of cells with protease inhibitor and phosphatase inhibitor. The obtained lysate was centrifuged at 16,000 rpm for 20 min, the supernatant was collected and protein levels were estimated. 50 ug protein was separated using SDS-PAGE (10%) electrophoresis, then transferred onto PVDF (Polyvinylidene difluoride membrane) membrane. The membrane was blocked using 3% BSA in 1X TBST for 2 h. The membrane was washed three times, 10 min each using TBST then incubated with the primary antibodies at 4 °C overnight, followed by incubation with horseradish peroxidase-conjugated anti-IgG secondary antibodies for two hours. The blots were detected using ECL solution (Westar Antares, Cyanagen, Bologna, Italy) in Syngene GBox Chemi XRQ gel documentation system. Quantification of protein band intensity was done using ImageJ software and relative density was calculated in comparison to alpha-tubulin expression.

Protein was extracted from livers, Raw 264.7 cells, primary hepatocytes, and primary NPCs by homogenization with T-PER buffer. After electrophoresis, gels were blotted to a PVDF membrane, and the membrane was blocked and incubated with primary antibodies. After overnight incubation, the membranes were washed and incubated with secondary antibodies (Goat anti-Rabbit IgG HRP; Catalog # 31460, Goat anti-Mouse IgG HRP; Catalog # 31430, Thermo Fisher Scientific, MA, USA). The bands were observed with ECL solution (XLS025-0000, Cyanagen, BO, Italy) after washing three times.
The following primary antibodies were used: β-actin (sc-130656, Santa Cruz Biotechnology), phosphor-IκBα (pIκBα), NF-κB p65, phosphor-NF-κB p65 (pNF-κB p65) (9936, CST, MA, USA), EGFR (A2909, ABclonal, MA, USA), phosphor-EGFR (9789, CST, MA, USA), HMGB1 (CSB-PA01604A0Rb, Cusabio, TX, USA), PGRMC1 (rabbit monoclonal, 13856, CST, MA, USA), and IκBα (mouse monoclonal, 9936, CST, MA, USA).