Chemiluminescence imaging system

Colon cancer cells were added with IC261 alone or combination with 2-DG. After cells were cultured with 20h, the total protein was extracted. And then protein concentrations were determined using the BCA protein assay (Beyotime, China). For western blotting analysis, equivalent amounts of protein (30 μg) were separated by 10% (w/v) SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Bio-Rad, CA). The membranes were blocked with 5% (w/v) skim milk in buffer for 2h at room temperature and then incubated with primary antibodies (cleaved caspase-3, p53, TIGAR, G6PD were purchased from Cell Signaling Technology USA; Tublin was purchased from Sigma USA) overnight at 4℃, followed by incubation with secondary antibodies (Abm, China) at room temperature for 1h. Data were collected using a chemiluminescence imaging system (vilber, France) and image J softwire for quantitative analysis.

The samples were characterized by Western blot (WB) analysis. LV20 VLPs samples were subjected to 15% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane by a transfer device. Western Blot analysis was performed using anti-M1 monoclonal rabbit antibody or anti-MTP polyclonal mouse antibody as primary antibody. Alkaline phosphatase-conjugated anti-rabbit or anti-mouse IgG (ImmunoWay, Plano, TX, USA) was used as the secondary antibody. Proteins were then stained using a chromogenic kit (Promega Corporation, Madison, WI, USA). The results were analyzed using a chemiluminescence imaging system (Vilber Lourmat, Marne-la-Vallée, France).