β actin protein

Samples were separated by SDS-PAGE on a 10% acrylamide gel and blotted on to a PVDF membrane (Millipore, Hellerup, Denmark), blocked in 3% fish gel (Invitrogen, Paisley, UK) in Tris buffered saline with 0.05% tween 20 (pH = 7.4) and incubated overnight in primary antibody recognising either UCP1 protein (AB 10983, Abcam, Cambridge, UK), phosphorylated STAT3 protein (#9145,Cell Signaling, Danvers, MA, USA), STAT3 protein (#9139, Cell Signaling) or β-actin protein (#4967, Cell Signalling). The following day, membranes were incubated in horseradish peroxidase-conjugated polyclonal anti-rabbit secondary antibodies (Dako, Glostrup, Denmark) in 3% fish gel. Bands at ∼33 kDa (UCP1), ∼79 kDa (STAT3) and ∼45 kDa (β-actin) were visualized using luminescence on a Carestream IS 4000IM and quantified using Carestream Health Molecular Imaging software (Fisher Scientific, ThermoFisher Scientific, Waltham, MA, USA).

HepG2 cells were seeded in 6-well plate at the density of 1 × 10⁵ cells/well for 24 h. Then, the cells were treated with casearlucin A for 36 h, the cells were washed with cold PBS twice and collected. The cells were lysed with lysis buffer containing protease inhibitor cocktail and PMSF. Then, the lysates were centrifuged at 10,000 rpm for 10 min and the supernatants were collected to acquire the total protein. Protein concentrations were quantified using the BCA protein assay kit (Beyotime, P0012S). The proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride. The membrane was blocked with 5% skim milk for 1 h at room temperature and then incubated (4°C, overnight) with primary antibodies against Bax (Cell Signaling Technology, 14796S), Bcl-2 (Cell Signaling Technology, 4223S), FAK (Cell Signaling Technology, 3285S), p-FAK (phospho Y397) (Cell Signaling Technology, 8556S), and MMP-2 (Cell Signaling Technology, 87809S). The membranes were subsequently incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Lastly, the protein blots were visualized using an ECL detection kit (Beyotime, P0018AS). β-Actin protein (Cell Signaling Technology, 4970S)was used as an internal reference. Each band was quantified by Image J software (20 (link)).