Polyvinylidene di uoride pvdf membranes

The following antibodies were used: NeuN (Millipore Biotechnology, MAB377); GPER (sc-48525); Iba1 (sc-32725), GAPDH (sc-32233), NLRP3 (Life science, Lot L27693), NLRP3 (ab4207), ASC (sc-22514-R), CD 11b (Gentex, GTX76060 ), Cleaved caspase 1 (Cell signaling, #4199S ), Cleaved IL1β (sc-7884), IL1RA (ab124962), and IL1RA blocking peptide (ab200257), NFkB-p65 (ab32568), H2A (ab177308), Bcl2 (sc-492), Cleaved caspase 3 (D175, 5A1E), CREB (Cell signaling, #9197X), p-CREB (Cell signaling, #9198S), Tubulin (sc-9104). Alexa-conjugated secondary antibodies were from Molecular Probes/Invitrogen (Carlsbad, CA). Polyvinylidene di uoride (PVDF) membranes with pore size of 0.45 µm were from Millipore (USA). BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and NBT (nitro blue tetrazolium) were from Promega (Madison, WI). TUNEL Kits (LOT #1639496, Life technologies) were from Active Motif Company. Duolink PLA kits (DUO92105), Duolink PLA Rabbit MINUS (DUO92003) and PLA Goat PLUS (DUO92005) proximity probes were from SIGMA-ALDRICH. Unless indicated otherwise, all the other chemicals were from Sigma-Aldrich (St. Louis, MO).

The experimental minks were euthanized and the brain tissues were aseptically harvested. The brain tissues were subsequently treated by liquid nitrogen and lysed the brain tissue in RIPA lysis buffer (Beyotime Biotechnology, China) for 30 min. Then transferred to a centrifuge tube and centrifuged at 12,000 g for 10 min at 4˚C.Then collected the supernatant and using BCA protein assay kit (Pierce, USA) to determine protein concentration. Total proteins were separated by 10% SDS-PAGE electrophoresis and transferred onto polyvinylidenedi uoride (PVDF) membranes (Millipore Corporation, USA). The membranes were then blocked (4˚C). Next, the membranes were incubated with the primary antibody overnight (4˚C), followed by secondary antibodies HRP-conjugated for 1.5 h (37˚C). Then using PBST washes the membranes. The protein expression of p-CREB, CBP30 and p300 were detected with the Gel Imaging System and the Quantity One Software version 4.0 (Bio-Rad, USA). Band intensity levels were normalized to β-actin (Sigma-Aldrich, USA). The information of the antibodies was described in Supplemental Table 3.