0.2 μm polyvinylidene difluoride pvdf membranes

After treatment with decided condition, the A549 lung cancer cells were collected and incubated with RIPA lysis buffer which contains NaCl 150 mM, Tris-HCl pH 7.6 25 mM, 1% sodium deoxycholate, 1% NP-40, 0.1% SDS for 30 min at 4 °C. The lysates were collected and their protein content were determined using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA). An equivalent number of proteins from each sample were separated by SDS-PAGE and transferred to 0.2 μm polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The separating blots were blocked with 5% skim milk in TBST (Tris-buffer saline with 0.1% tween containing NaCl 125 mM, Tris-HCl pH 7.5 25 mM and 0.1% tween 20) for 2 h and incubated with primary antibody overnight at 4°C. Secondary antibodies were incubated for 2 h at room temperature after being washed by TBST three times. Finally, the protein bands were detected using chemiluminescence substrate and exposed by Chemiluminescent ImageQuant LAS4000. Protein bands were analyzed using ImageJ software (version 1.52, National Institutes of Health, Bethesda, MD, USA).

On day 8 after the initiation of differentiation treated with various concentrations of ABB (0–25 μg/mL), cells were rinsed three times with cold PBS and scraped into lysis buffer (20 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol, 2% Nonidet P-40, 1 mM EDTA, 20 mM sodium fluoride, 30 mM sodium pyrophosphate, 0.2% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), and 1 mM sodium vanadate). The cell lysate was centrifuged for 20 min at 12,000 ×g and the supernatant was collected. Each protein extract (5, 10, or 20 μg) was loaded on 10% SDS-polyacrylamide gel and the separated proteins were blotted onto 0.2 μm polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA) using a semidry transfer apparatus (Trans-Blot SD; Bio-Rad). The membranes were then incubated with primary antibody (Akt-p [1 : 1000], Akt [1 : 2000], perilipin [1 : 2000], and actin [1 : 1000]) diluted in blocking solution at 4°C overnight. Membranes were probed with horseradish peroxidase-labeled secondary antibody (anti-goat IgG or anti-rabbit IgG) for 1 hour at room temperature. After several washing steps, the immunoreactive bands were detected by chemiluminescence using West-one (iNtRON Biotechnology).