Anti p ripk3

Total proteins of adipose tissues were extracted and western blot was carried out as previously described^{51 (link)}. The following antibodies were used: anti-β-Actin (1:1000, Protein Tech, 60008-1-Ig), anti-MED20 (1;1000, Protein Tech, 17598-1-AP), anti-GAPDH (1:5000, CST, 5174 s), anti-MLKL (1:1000, Abcam, ab184718), anti-P-MLKL (1:1000; Abcam, ab187091), anti-Cleaved Caspase-3 (1:1000, CST, 4190), anti- Acetyl-CoA Carboxylase1 (1:1000, CST, 4190), anti-ACLY (1:1000, CST, 13390), anti- RIPK3 (1:1000, ABclonal, A5431), anti-RIPK1 (1:1000, ABclonal, A7414), anti-P-RIPK3 (1:1000, Abcam, ab205421), anti-P-RIPK1 (1:1000, ABclonal, AP1230), anti-C/EBPα (1:1000, CST, 8178 S), anti-PPARγ (1:1000, Santa Cruz, sc-7273), anti-CD36 (1:1000, Sino Biological Inc, 80263-T48), anti-Perilipin 1 (1:1000, CST, 9349 s) and anti-FASN(1:1000, CST, 3180).

Fresh pancreatic tissues were homogenized in RIPA buffer (Nacalai Tesque, Kyoto, Japan) containing phosphatase inhibitor cocktail (Roche, Basel, Switzerland), protease inhibitor cocktail (Roche, Mannheim, Germany), and 1 mmol/L phenylmethylsulfonyl fluoride (Sigma, St. Louis, USA). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Scientific, no. 23225). Total protein was denatured in a loading buffer containing SDS at 95 °C for 5 min. Fifty micrograms of denatured protein was loaded and separated on a sodium dodecyl-sulfate polyacrylamide gel electrophoresis gel and transferred to a polyvinylidene difluoride membrane. Membranes were probed with the following antibodies: anti-pRIPK3 (#ab195117, Abcam) and beta-actin (#sc-47778, Santa Cruz). All primary antibodies were used at a dilution of 1:1000. Anti-rabbit IgG horseradish peroxidase and anti-mouse-IgG horseradish peroxidase secondary antibodies were used. All secondary antibodies were used at a dilution of 1:10,000. Detection of the immune complexes was attained by using ECL western blotting detection reagent (Amersham^TM).