Duolink pla rabbit minus

SCC15 or SCC-QLL1 human HNC cells were attached on glass cover slips (Corning, Lowell, MA) at 70% confluence and cultured overnight. On the next day, cells were washed with PBS, treated with NTP, and cultured for an additional 24 hours in the absence of serum. The cells were fixed in 4% paraformaldehyde for 15 min, permeablized with 0.1% Triton X-100, and incubated with 5% BSA for 1 hour at room temperature. Cells were incubated with rabbit anti-MUL1 (1:200) and mouse anti-pan-AKT (1:200) antibodies at 4°C overnight. After washing, the slides were incubated with Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity probes, and proximity ligation was performed using the Duolink^® with PLA^® Technology reagent (Sigma, St. Louis, MO) according to the manufacturer's protocol. Fluorescence was detected using an EVOS FL AUTO microscope (Life Technology, NY, USA).

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); and NF-κB-p65 (ab32568), H2A (ab177308), Bcl2 (sc-492), cleaved caspase 3 (D175, 5A1E), CREB (cell signaling, #9197X), and p-CREB (cell signaling, #9198S), tubulin (sc-9104). Alexa-conjugated secondary antibodies were from Molecular Probes/Invitrogen (Carlsbad, CA). Polyvinylidene difluoride (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).