Pierce ecl advance western blotting detection reagents

The RAW 264.7 cells were seeded in 6-well plate at the density of 2 × 10⁶ cells/well for 24 h. Cells were treated with the presence or absence of KM1608 at the concentration of 100 µg/mL. After multiple time-points, cells were collected and washed with Dulbecco's phosphate-buffered saline (DPBS), before lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with 1X protease inhibitor cocktail and 1 mM sodium orthovanadate (Na₃VO₄) phosphatase inhibitor to get the whole-cell extracts according to the manufacturer’s instructions. Protein concentration of each whole-cell extracts was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific). The equal protein amounts of each whole-cell extracts (10 μg/lane) were separated by electrophoresis in a 10% sodium dodecyl sulfate-polyacrylamide gel and blotted onto PVDF transfer membranes. Epitope-specific primary antibodies included phospho-c-Jun NH2-terminal kinase (p-JNK), phospho-extracellular signal–regulated kinase (p-ERK), and β-actin conjugated with secondary antibodies (Cell Signaling, Boston, MA, USA) were used to label the target proteins. The bound antibodies were detected by PierceTM ECL Advance Western Blotting Detection Reagents (Thermo Scientific) and visualized with FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Germany).

HT22 cells were seeded into 6-well plates at 2 × 10⁵ cells/well for 24 h and then treated with JP-203 at 25 and 50 μM in the presence or absence of 5 mM glutamate. After 6 h, cell pellets were collected and washed with DPBS. RIPA buffer supplemented with protease inhibitor cocktail (1X) was added to obtain whole-cell protein extracts according to the manufacturer’s instructions. The protein concentration of the cell extracts was evaluated using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA).
Each 10 μg of extracted samples was separated by SDS-PAGE and blotted onto PVDF transfer membranes. The primary antibodies included HO-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cell signaling Technology, Danvers, MA, USA) and horseradish peroxidase (HRP)-conjugated secondary antibodies (Cell Signaling, USA) were used to label the target proteins. The blots were developed using PierceTM ECL Advance Western Blotting Detection Reagents (Thermo Scientific, Waltham, MA, USA) and detected with FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany).

3T3-L1 cells were seeded in a 6-well plate at a density of 1 × 10⁶ cells/well for 24 h and treated with compound 1 at indicated concentrations (0, 25, 50, 100 µM). After 24 h, cells were collected and lysed in RIPA buffer (Tech&Innovation, Gangwon, Republic of Korea) following the manufacturer’s instructions to obtain whole-cell extracts. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). The proteins separated by electrophoresis were transferred onto PVDF membranes. Primary antibodies, including PPARα, FAS, adiponectin, CPT1A, and β-actin, were used in combination with conjugated secondary antibodies (Cell Signaling, Boston, MA, USA) to label the target proteins. The bound antibodies were detected using Pierce™ ECL Advance Western Blotting Detection Reagents (Thermo Scientific, Waltham, MA, USA) and visualized with the FUSION Solo Chemiluminescence System (PEQLAB Biotechnologie GmbH, Erlangen, Germany).