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).