Rabbit anti rip3 ab

For immunoblotting analysis, electrophoresis of kidney cell lysates or cell lysates from the NZM2328 podocyte cell line was conducted with 10% running gels and 5% stacking gels. Proteins from the cell lysates were then transferred onto a polyvinylidene fluoride membrane. After blockade with 5% non-fat dry milk in Tris buffered saline with 0.1% Tween 20 (TBST), membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-RIP3 Ab (Abcam), anti-p-RIP3 Ab (Abcam), anti-MLKL (Abcam), anti-p-MLKL Ab (Abcam), mouse anti-NLRP3 (Adipogen), anti-caspase-1 p20 (Adipogen), rabbit anti-caspase-8 (Abcam) and anti-GAPDH Ab (Cell Signaling Technology). After washing 3 times with TBST, membranes were incubated with their corresponding secondary antibodies. The signals on the membranes were detected by a chemiluminescence analysis kit (Millipore).

Stimulated podocytes were washed 3 times with PBS and blocked for 1 hour with 5% BSA in PBS. Then the cells were incubated with mouse anti-RIP1 Ab (Abcam) and rabbit anti-RIP3 Ab (Abcam) or rabbit anti-RIP3 Ab and mouse anti-NLRP3 Ab (Adipogen) at 4°C overnight. The RIP1-RIP3 interactions and RIP3-NLRP3 interactions were detected using the PLA kit (Duolink, Sigma) according to manufacturer’s protocol (16 (link)). Podocytes were labeled with anti-mouse synaptopodin Ab (Bioss) and stained with DAPI. Mounting media (Vector) was used. A Zeiss LSM 800 confocal microscope was used for analysis.