Rabbit anti tlr4 antibody

After removing the culture medium, cancer cells were washed twice with ice-cold PBS. The harvested cells were then lysed in a lysis buffer containing protease inhibitor and a phosphatase inhibitor cocktail (Roche Mannheim, Mannheim, Germany) for 30 min at 4 °C and centrifuged at 12,000 rpm for 15 min. The supernatants were collected and the protein concentrations were measured using a Pierce BCA Protein Assay Kit (Thermo Fischer Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Equal quantities of total proteins were separated on a 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). The membranes were blocked in 5% nonfat milk for 1 h and then incubated overnight with primary antibody including rabbit anti-MOR antibody (1:1,000; Abcam, Cambridge, MA, USA), rabbit anti-TLR4 antibody (1:500; Abcam), rabbit anti-PD-L1 antibody (1:500; Abcam), and rabbit antibodies to p-ERK, ERK, p-JNK, JNK, p-p38, p38, p-Akt, Akt, p-P65, and P65 (1:1,000; Cell Signaling Technology, Danvers, MA, USA). The PVDF membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. The blots were developed using the ECL substrate (Amersham, Aldermaston, UK). Glyceraldehye 3-phosphate dehydrogenase (1:2,000; Abcam) served as the endogenous control for the protein quantification.