Ab217798

After 48 h of incubation, cells were washed by cold PBS three times and lysed on ice with whole cell lysate for 10 min. Proteins contained were quantitated by BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). A measure of 30 µg of total proteins were then separated by polyacrylamide gel electrophoresis (PAGE) at a constant voltage of 80 V for 35 min followed by 120 V for 45 min, and sequentially transferred onto the polyvinylidene fluoride (PVDF) membranes (Amersham, USA) after electrophoresis. The membranes were blocked with 5% skim milk at room temperature for 1 h. Afterwards, the membranes were incubated overnight at 4 ℃ with primary rabbit polyclonal antibodies containing ANGPT2 (ab155106, 1:1000, abcam, Cambridge, UK), NOD1 (ab217798, 1:1000, abcam), NOD2 (ab36836, 1:1000, abcam), NF-κB (ab194729, 1:1000, abcam) and GAPDH (ab9485, 1:2500, abcam). Then, the membranes were incubated with secondary antibody, horse radish peroxidase-labeled goat anti-rabbit IgG H&L (HRP) (ab6721, 1:2000, abcam) for 1 h. Protein bands were visualized under the optical luminescence instrument (GE, USA), and were quantified by gray scale scanning using the Image Pro Plus 6.0 software (Media Cybernetics, USA).

The whole cell lysates (Beyotime, Shanghai, China) were used to harvest total protein. The equal amount of protein extract was then separated by SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membrane. Subsequently, membranes were blocked in 5% non-fat dry milk. The membranes were incubated with the appropriate dilutions of primary antibodies against NOD1 antibody (1:1000, ab217798, Abcam, Cambridge, MA, US), BAK1 (1:1000, ab32371, Abcam, Cambridge, MA, US), NLRP6 (1:1000, ab58705, Abcam, Cambridge, MA, US), and GAPDH (1;1500; Abcam, Cambridge, MA, USA) antibody sampler kit (9782 T) at 4 °C. The more detailed steps have been illustrated as previously described [24 (link)].