Pvdf membrane 0.45μm

For the immunoblot analyses, the total protein was extracted from the cytoplasm of the IPEC J2 cells, and the samples were denatured in 4×SDS-PAGE loading buffer (40 mM Tris-HCl, PH 8.0, 200 mM DTT, 4% (v/v) SDS, 40% (v/v) Glycerol and 0.032% (v/v) Bromophenol Blue) (No. 7173 Takara, Ostu, Japan) and boiled for 10 min. The denatured proteins were separated using 12% SDS-PAGE and were transferred onto a PVDF membrane (0.45 μM) (Millipore, Boston, Massachusetts, USA). The membrane was blocked in Tris Buffered Saline with Tween (TBST) (10 mM Tris, 100 mM NaCl, 0.1% Tween 20) with 5% (w/v) nonfat milk powder for 1.5 h. After washing with TBST for three times, the blocked membranes were incubated with the primary antibodies overnight at 4°C in TBST and were then washed three times followed by incubation with the corresponding HRP-linked secondary antibodies (1:2500) for 1 h at room temperature. The membranes were washed three times, and bound antibodies were detected using an ECL plus detection system (P1010, Applygen, Beijing, China). The expression of each protein was normalized to that of β-actin.

Around 20 μg of each sample was separated by 10% SDS-PAGE gels and transferred to a polyvinylidine fluoride fluoropolymer (PVDF) membrane (0.45 μm, Millipore, Darmstadt, Germany) using Trans-Blot Turbo transfer system (Bio-Rad, California, CA, USA). The membrane was blocked with TBST (10 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20, pH 8.0) containing 5% BSA at 4 °C overnight. The target protein bands were sequentially detected by acetyl lysine primary antibodies (1:1000 dilution in TBST) (ImmuneChem, Burnaby, BC, Canada) and HRP (horseradish peroxidase–conjugated) secondary antibodies (1:1000 dilution in TBST) (Beyotime Company, Shanghai, China), and finally visualized with enhanced chemiluminescence reagent (Thermo, Waltham, MA, USA).

MCF-7 and HepG2 tumor cells were cultured overnight in opti-MEM medium and pre-treated with E5 (5 μM) and M-E5 (E5: 5 μM, PEG-PE: 20 μM) for 1 h at 37°C, followed by treatment with CXCL12 (200 ng/ml) for another 30 min. Later on, cells were harvested, washed, and lysed in ice cold RIPA lysis buffer containing a mixture of protease inhibitor and phosphatase inhibitor for 30 min. The debris was removed by centrifugation at 14,000 x g for 10 min. Equal amount of proteins (20 μg) from each sample was loaded and separated on 10% polyacrylamide gel and transferred to a PVDF membrane (0.45 μm; Millipore, Bedford, MA) by semidry transfer (Trans-Blot SD Semi-dry Transfer cell, Bio-Rad, USA). The membrane was blocked with 5% (w/v) BSA in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature. Later on the membrane was treated with primary antibodies (Erk, phosphorylated-Erk, Akt, phosphorylated-Akt, p38, phosphorylated-p38, GAPDH) overnight at 4°C followed by incubation with the appropriate peroxidase-conjugated secondary antibody for 1 h at room temperature. The membrane was washed with TBST for three times and then developed with enhanced chemiluminescence system.

In brief, around 20 μg of each extracted total proteins was separated by 10% SDS–polyacrylamide gels, and subsequently transferred to a polyvinylidine fluoride fluoropolymer (PVDF) membrane (0.45 μm, Millipore, Darmstadt, Germany) using a Trans-Blot Turbo transfer system (Bio-Rad, Hercules, CA, USA). The TBST (10 mM Tris-HCl, 150 mM NaCl, and 0.05% Tween 20, pH 8.0) containing 5% BSA was used to block the transferred membrane for overnight at 4 °C. Acetyl lysine primary antibodies (1:1000 dilution in TBST) (ImmuneChem, Burnaby, BC, Canada) and secondary antibodies HRP (horseradish peroxidase–conjugated) (1:1000 dilution in TBST) (Beyotime Company, Shanghai, China) were used to detect the target protein bands, and lastly, visualized using the enhanced chemiluminescence (Pierce, Waltham, WA, USA).

RPE cells (2.5 × 10⁵/well, 6-well plate) were seeded and cultured for 3 days. Before treatment with TNF-α and/or AICAR, cells were serum-starved for 24 hours in serum-free medium. After treatment, cells were washed with cold PBS and lysed in NP40 cell lysis buffer (Invitrogen, Carlsbad, CA) containing protease and phosphatase inhibitor cocktail (Roche, Indianapolis, IN). Samples were loaded onto a NuPAGE 4–12% Bis-Tris Gel (Novex, Carlsbad, CA), transferred to a polyvinylidene difluoride (PVDF) membrane (0.45 μm; Millipore, Billerica, MA), blocked with 5% non-fat dry milk, and incubated with appropriate primary antibodies. Blots were subsequently incubated with secondary antibodies and images were developed using chemiluminescent substrate (ECL Select western blotting detection reagents, GE Healthcare Life Sciences, Piscataway, NJ). Band signals were detected by an image-scanning densitometer (ChemiDoc imaging system; Bio-Rad) and quantitated by ImageJ 2.0.