Enhanced chemiluminescence western blot detection system

After treatment, the cells were collected and lysed at 4 °C in lysis buffer containing protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The whole cell lysate was centrifuged at 4°C, and the supernatant was separated by SDS-PAGE and electrotransferred to a PVDF membrane (Millipore, Billerica, MA). The membranes were incubated at 4 °C overnight in blocking buffer, and they were further incubated for 2 h at room temperature with specific primary antibody in blocking buffer. After three washes in PBS with 0.1% Tween 20, the membrane was incubated for 1 h at room temperature with HRP-conjugated secondary antibody in blocking buffer and developed through an enhanced chemiluminescence Western blot detection system (Millipore, Billerica, MA).

Cells were lysed using cell lysis buffer (Beyotime Institute of Biotechnology) with Protease Inhibitor Cocktail (100X; Sangon Biotech Co., Ltd., Shanghai, China) and 1 mM phenylmethylsulfonyl fluoride (Sigma-Aldrich; Merck KGaA). Following centrifugation at 4°C and 12,000 × g for 15 min, protein concentration in the supernatant of the lysates was determined using the Bradford Coomassie Blue G-250 method. Proteins (40 µg per lane) were separated using 10% SDS-PAGE, and transferred onto polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA) in sequence. Thereafter, PVDF membranes were blocked with 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) in TBS containing 0.05% Tween-20 at room temperature for 2 h. Subsequently, PVDF membranes were incubated with primary antibodies at 1:1,000 in TBS containing 0.05% Tween-20 and 5% bovine serum albumin overnight at 4°C for 16 h. Next, PVDF membranes were incubated with corresponding HRP-conjugated secondary antibodies (1:1,000) at room temperature for 2 h. Finally, membranes were visualized using an enhanced chemiluminescence Western blot detection system (EMD Millipore). In the present study, all relative protein concentrations were normalized to that of β-actin.

The cells were collected at the end of each experiment, then washed three times with pre-cooled PBS. Proteins were extracted from the cells using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China). The protein concentration was determined using the 2-D Quant kit (GE Healthcare Life Sciences, Chalfont, UK) method. The samples (30 µg/lane) were separated on a 10% SDS-PAGE, and then transferred onto a PVDF membrane. Subsequently, the membrane was blocked with 5% non-fat milk for 1 h at room temperature. The membrane was incubated with antibodies against β-actin, PCSK9, Bax, caspase-3, Bcl-2, p38, p-p38, ERK, p-ERK, JNK and p-JNK at 4°C overnight (Table II). Following the overnight incubation, the membrane was washed three times with TBS-Tween-20 (TBST) for 30 min, and the membrane was incubated with the secondary antibodies (Abcam) for an additional 1 h at room temperature. The membrane was washed three times with TBST for 30 min and visualized using the enhanced chemiluminescence Western blot detection system (EMD Millipore, Billerica, MA, USA). Reference gene for the western blot analysis was β-actin. All antibody details are listed in Table II.

Firstly, murine B16 melanoma cells were lysed using a lysis buffer (Beyotime Institute of Biotechnology, Haimen, China). Lysate concentration were measured using a Bradford assay. Following centrifugation at 4,800 × g for 5 min at 4°C, the lysates were boiled in sodium dodecyl sulfate (SDS) loading buffer for 5 min. Proteins (50 µg) were separated by 10% SDS-PAGE, and then transferred to a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membrane was blocked with 5% Tris-buffered saline (TBS), 0.3% Tween-20, 5% non-fat milk for 2 h at 37°C, and incubated at 4°C overnight with primary antibody of rabbit polyclonal anti-p38 (1:5,000; catalog no. 9212; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).
Following washing with 0.1% Tween-TBS three times, the membrane was further incubated with the horseradish peroxidase conjugated secondary antibody goat anti-rabbit IgG-H&L (1:10,000; catalog no. A21076, Bioworld Technology, Inc., St. Louis Park, MN, USA) for 1 h at 25°C. Protein signals were visualized by using the enhanced chemiluminescence western blot detection system (EMD Millipore), and β-actin was used as the internal control.