Myd88

Whole-cell protein lysates were solubilized in 100-200 μl of radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) supplemented with a 1% protease inhibitor cocktail (Beyotime, China) for 20 min on ice. The protein concentration was determined using a BCA protein assay kit (Beyotime, China). The proteins (30 μg) were denatured and separated on an 8%-15% discontinuous SDS-polyacrylamide gel (SDS-PAGE) by electrophoresis and subsequently electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane. After blocking with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h at room temperature, the PVDF membrane was incubated overnight at 4 °C with the appropriate primary antibody for TLR7 (1:100; Novusbio, USA), MyD88 (1:200; Boster, China), IRF7 (1:1000; Abcam, USA), beclin 1 (1:200; Boster, China), SQSTM1 (1:200; Boster, China), EV71/CA16-VP1 (1:1000; Millipore, USA) or GAPDH (as a loading control, 1:10000; Abmart, China). Then, the membrane was extensively washed three times with TBST prior to incubation for 1 h at room temperature with the corresponding secondary antibody (Abmart, USA) at a dilution of 1:12,000. Finally, the membrane was again extensively washed three times with TBST and then visualized using enhanced chemiluminescence reagents (Beyotime, China) and X-ray films (Kodak, Japan).

MCF-7 and MDA-MB-231 cells in LPS group were treated with 2 µg/ml LPS for 48 h. In TLR4 blocking group, 100 nmol/L eritoran was added to the cell culture for 30 min prior to the addition of 2 µg/ml LPS for 48 h. For western blot analysis, 1×10⁶ cells were lysed in modified RIPA buffer (Beyotime, Shanghai, China). The protein concentration was measured by BCA Protein Assay Kit (Beyotime) and samples were loaded on 10% SDS-PAGE. Following electrophoresis, the proteins were transferred to a BioTrace PVDF membrane (Pall Life Sciences, Ann Arbor, MI, USA) and blocked in 5% non-fat dried milk for 1 h at room temperature and incubated with rabbit antibodies against TLR4 (1∶1000; Abcam, Cambridge, MA, USA), MyD88 (1∶500, Boster, Wuhan, China) and GAPDH (1∶5000, Abcam, Cambridge, MA, USA) overnight at 4°C. After washing three times with Tris-buffered saline Tween 20 (TBST), membranes were incubated with anti-rat IgG (1∶5000, Bioworld, Dublin, OH, USA). The membrane was washed three times with TBST, and signals were detected by enhanced chemiluminescence system (Amersham Pharmacia Biotech, Bucks, UK).