Western blotting
Total protein was extracted from fresh-frozen intestinal biopsies and pWSLV-02-LILRA3 plasmid- or pWSLV-02-transfected U937 cells using a mixture of RIPA lysis solution containing protease inhibitor and phosphatase inhibitor. The protein concentration was determined using a BCA assay kit. RIPA and the BCA kit were purchased from Beyotime Biotechnology (Shanghai, China). A total of 40 µg protein from each sample was separated by 12.5% or 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, USA). The membranes were blocked with 5% nonfat milk in TBST (Tris-buffered saline (TBS) with 0.1% Tween 20) at room temperature for 1 hour and then incubated with specific primary antibodies in TBST overnight at 4°C followed by three washes with TBST. The membranes were then incubated with the following antibodies: anti-LILRA3 polyclonal antibody (pAb) (Cat. GTX108819, GeneTex, Alton Pkwy Irvine, USA); anti-CD36 pAb (Cat. 18836-1-AP) and anti-CD206 pAb (Cat. 18704-1-AP) were obtained from Proteintech (Wuhan, China); anti-p-Foxo3a mAb (Cat. ab47285) and anti-Foxo3a mAb (Cat. ab17026) were procured from Abcam (Cambridge, USA); anti-p-Akt monoclonal antibody (mAb) (Cat. #4060), anti-Akt mAb (Cat. #4691), anti-p-MEK1/2 mAb (Cat. #9154), anti-MEK1/2 mAb (Cat. #8727), anti-p-Erk1/2 mAb (Cat. #4370), anti-Erk1/2 mAb (Cat. #4695), anti-p-P38 MAPK mAb (Cat. #4511), anti-P38 MAPK mAb (Cat. #8690), anti-p-PDK1 mAb (Cat. #3438), anti-PDK1 pAb (Cat. #3062), anti-p-c-Raf pAb (Cat. #9421), anti-p-PI3K pAb (Cat. #4228), anti-PI3K mAb (Cat. #4257), anti-p-P65 mAb (Cat. #3033), anti-P65 mAb (Cat. #8242), anti-p-SAPK/JNK mAb (Cat. #4668), anti-JNK2 mAb (Cat. #9258), anti-p-mTOR mAb (Cat. #5536), anti-mTOR mAb (Cat. #2983) were purchased from Cell Signaling Technology (CST, Danvers, USA). GAPDH (Cat. KM9002, Sungene Biotechnology, Tianjin, China) was used as the internal reference. The membranes were then incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse antibody (Cat. LK2001/LK2003, Sungene Biotechnology, Tianjin, China) in TBST for 2 hours at room temperature. After 3 washes with TBST, the blots were visualized using enhanced chemiluminescence kit (Bio-rad, Hercules, USA).