Il 1β af 401 na

CIP (P4978) and DMSO were purchased from Sigma-Aldrich. PKCα (C2-4) inhibitor peptide (17478) was purchased from Cayman Chemical. Flag-PKCα, -β, -δ, and -ξ plasmids were a generous gift from Dr. D. Zhou (Xiamen University, China). The GST-tagged GRA7 and truncated mutant genes were described previously [17 (link)]. V5-tagged AC or AU1-PLD1 and truncated mutant genes were cloned into the XbaI and BamHI sites in pcDNA3.0. All constructs were sequenced using an ABI PRISM 377 automatic DNA sequencer to verify 100% correspondence with the original sequence. Specific antibodies against phospho-(Thr147)-PLD1 (3831), phospho-(Ser561)-PLD2 (3834), PLD1 (3832), PLD2 (13904), PKCα (2056), PKCγ (43806), and NLRP4 (12421) were purchased from Cell Signaling Technology. Antibodies specific for actin (I-19), ASC (N-15-R), IL-18 (H-173-Y), TRAF6 (H-274), caspase-1 p10 (M-20), Rab5 (D-11), Rab7 (H-50), LAMP1 (E-5), LAMP2 (H4B4), Tubulin (B-5-1-2), Calnexin (H-70), FACL4 (N-18), VDAC (B-6), His (His17), V5 (C-9), Flag (D-8), and GST (B-14) were purchased from Santa Cruz Biotechnology. AU1 (GTX23402) and PKCβI (A10-F) were purchased from GenenTex and Antibodies-online Inc., respectively. IL-1β (AF-401-NA) and NLRP3 (AG-20B-0014) were from R&D Systems and Adipogen, respectively.

Cells were lysed in RIPA buffer supplemented with 1mM PMSF and 1× Complete Protease Inhibitor Cocktail (Roche). Lysates were spun at max speed in an Eppendorf microfuge at 4°C for 20 min and supernatants were mixed with 6× Laemmli sample buffer. To detect full length and FIIND processed NLRP1B, lysates were incubated at room temperature for 15min prior to SDS-PAGE. To analyze all other proteins, including the N-terminally cleaved form of NLRP1B, samples were boiled for 10min prior to separation. SDS-PAGE was performed with Novex 10% and 12% BisTris gel system according to manufacturer’s instructions (Invitrogen). Separated proteins were transferred to Immobilon-FL PVDF membranes. Membranes were blocked with Odyssey blocking buffer (Licor). The following antibodies were used for the following antigens: HA mAB 3F10 (Roche), MEK-2 SC-13115 (Santa Cruz), MYC mAb 9E10 (Clonetech), IL-1β AF-401-NA (R&D systems). Secondary antibodies anti-rat, mouse and goat were all conjugated to Alexa Fluor-680 (Invitrogen).

Menthol and bacterial lipopolysaccharide (LPS) (Escherichia coli, 0111: B4) were purchased from Sigma (USA). Mass spectrometry grade reagents (column, buffer et al.) were all purchased from Sigma (USA). The Mouse Proteome Profiler Array was purchased from R&D Systems (USA). The antibodies used were CCL3 (ab25128, Abcam, USA), IL-6 (12912; Cell Signaling Technology, USA), TNF-α (11948S; Cell Signaling Technology, USA), p-NF-κB (3033; Cell Signaling Technology, USA), NF-κB (8242; Cell Signaling Technology, USA), p-Akt (4051; Cell Signaling Technology, USA), Akt (9272; Cell Signaling Technology, USA), TLR1(2209; Cell Signaling Technology, USA), TNFAIP3 (5630; Cell Signaling Technology, USA), IFIT1 (14769; Cell Signaling Technology, USA), viperin (13996; Cell Signaling Technology, USA), IL-1β (AF-401-NA, R&D Systems, USA), and β-actin (A1978; Sigma-Aldrich, USA). HRP-conjugated secondary antibodies were obtained from Cell Signaling Technologies (7074, 7076; USA).