Anti flag f1804

Mouse tissues and cells were harvested and dissolved in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 1% Triton-X-100, 0.5% NP-40, pH 7.6) or NP-40 buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, pH 7.4) containing protease and phosphatase inhibitor cocktail (GenDEPOT, Huston). Immunoprecipitation was performed with anti-Flag (F1804, Sigma) antibody overnight at 4 °C, followed by addition to protein G-Sepharose beads (Upstate Biotechnology) for two hours at 4 °C. Liver nuclear extracts were prepared as described previously 27 (link). Protein lysates were performed to Western blotting with the following primary antibodies [rat anti-HA (3F10, Roche), rabbit anti-GLUT2 (ab54460, Abcam), rabbit anti-β-actin (AbC-2004, Abclon), mouse anti-Flag (F1804, Sigma), mouse anti-HSP90α/β (sc-13119, Santacruz), rabbit anti-CREBH (EWS101, Kerafast), mouse anti-Lamin B1 (sc-377001, Santacruz), goat anti-PTP4A1(EB06456, Everest), and rabbit anti-GSK3 β (#12456, Cell Signaling Technology)]. The membranes were incubated with primary antibodies followed by the horseradish peroxidase-conjugated secondary antibodies (rat: 31470, rabbit: 31464, Thermo Fischer Scientific; mouse: AbC-5001, AbClon) for one hour at room temperature. Immuno-reactive bands were visualized using a chemiluminescent substrate (RPN2106, GE).

Antibodies against the yeast Swr1 and H2A.Z were gifts of Carl Wu. The anti-FLAG (F1804) and the anti-H2A (39235) antibodies were purchased from Sigma-Aldrich and Active Motif (Carlsbad, CA), respectively. For H2A and H2A.Z western, both antibodies were used at a dilution of 1:2,000. ChIP-qPCR analysis was performed as previously described with the following modifications (Aparicio et al., 2005 (link)). ChIP reactions were performed using 1.25 mg dynabeads conjugated to Protein A or G. Five microliter of anti-Swr1 was used in each ChIP reaction. qPCR was performed on a LightCycler 96 Real-Time PCR system (Roche). The primers used in qPCR are listed in Supplementary file 2.

LPS (O111:B4, L2630), ATP (A7699), disuccinimidyl suberate (S1885), nigericin (N7143), and phorbol myristate acetate (PMA, P1585) were from Sigma-Aldrich; alum (77161) and the c-Myc tag (MA1-980) were from Thermo Fisher Scientific; and poly(dA:dT) was from InvivoGen. The following antibodies were used: anti-Flag (F1804) (Sigma); anti-mouse caspase-1 p20 (AG-20B-0042) and anti-mouse NLRP3 (AG-20B-0014) (AdipoGen); anti-human caspase-1 (ab108362) and anti-BRCC36 (ab108411) (Abcam); anti-mouse IL-1β (AF-401-NA) (R&D); anti-HA-tag (3724) (Cell Signaling Technology); and anti-VDR (sc-13133), anti-ASC (sc-514414), and anti-β-actin (sc-47778) (Santa Cruz). The Mouse TNF ELISA Kit (558534), Mouse IL-1β ELISA Kit (559603), and TMB Substrate Reagent Set (555214) were from BD Biosciences. The mouse IL-18 ELISA set (EMC06) was from ExCell Biotech (Table 1).

Western blotting was performed as previously described^{30 (link)}. Liver tissues and cultured cells were homogenized in ice-cold CelLytic MT Cell Lysis Reagent (Sigma) with protease inhibitors. Immunoprecipitation was performed using Acetylated K antibodies conjugated to Dynabeads Protein G (Life Technologies) or Flag tag antibody conjugated-magnetic beads. The antibodies used in the immunoprecipitation of Flag tag, acetylated K of GKRP, and acetylated K of keratin 8 were anti-DYKDDDDK tag magnetic beads (1E6, 10 μl/mg protein; Wako), anti-AcK (#9441, 0.2 μl/mg protein; Cell Signaling Technology, Danvers, MA), and anti-AcK (ab21623, 0.2 μl/mg protein; Abcam, Cambridge, UK), respectively. The following antibodies were obtained for immunoblotting: anti-Sirt2 (sc-20966, 1:500), anti-glucokinase (sc-7908, 1:500), anti-GKRP (sc-11416, 1:500) (Santa Cruz Biotechnology), anti-Sirt1 (07-131, 1:1000; Millipore, Billerica, MA), anti-Flag (F1804, 1:1000), anti-β-actin (A5441, 1:1000) (Sigma), anti-HA (3F10, 1:1000; Roche), anti-HAlo (G9281, 1:1000; Promega), and anti-keratin 8 (TROMA-I, 1:200; Developmental Studies Hybridoma Bank, Iowa City, IA). Immunoblot images were quantified by densitometry on an LAS-3000 Imager (Fujifilm, Tokyo, Japan). Uncropped images of representative immunoblots are shown in Supplementary Fig. 9.

HA-FLAG-USP13 was purchased from Addgene (Plasmid #22568, provided by Dr Wade Harper) and subcloned into pGEX-4 T-2 vector (Clontech). USP13 site mutants were generated by site-directed mutagenesis (Stratagene).
The anti-USP13 (GTX118595, dilution: 1:500) and anti-Rad51 (N1C2, dilution: 1:200) antibodies were purchased from Genetex. Anti-Ub (P4D1, dilution: 1:500), anti-RPA32 (9H8, dilution: 1:200) and anti-BRCA1 (D9, dilution: 1:200) antibodies were purchased from Santa Cruz Biotechnology. Anti-γH2AX (05-636, dilution: 1:500), anti-FK2 (04-263, dilution: 1:500) and anti-MDC1 (05-1572, dilution: 1:200) were purchased from Millipore. Anti-RAP80 (A303-763A, dilution: 1:500) and anti-53BP1 (A300-272A, dilution: 1:500) were purchased from Bethyl Laboratories. Anti-FLAG (F1804, dilution: 1:1,000), anti-HA (H9658, dilution: 1:1,000), and anti-β-actin (A1978, dilution: 1:2,000) antibodies were purchased from Sigma. Anti-RNF8 (ab4183, dilution: 1:500) was purchased from Abcam.

Anti-GAPDH (5174), anti-Cyclin D1 (2978) anti-TIP60 (12058), anti-P21 (2947), and anti-PCNA (13110) were from Cell Signaling Technology. Anti-Flag (F1804) was purchased from Sigma-Aldrich. Anti-NR2F6 (ab137496) was obtained from Abcam.