Total iκbα

Western-blotting was performed with precast gradient gels (Bio-Rad) using standard methods as described previously [36 (link), 37 (link)]. Briefly, total protein from each sample was resolved in 10% sodium dodecyl sulfate (SDS)-polyacrylimide gel electrophoresis and was transferred to the Immobilon™ PVDF Transfer Membranes (Millipore Corporation, Billerica, MA). The membrane was then blocked in 5% bovine serum albumin (BSA) and incubated with the primary antibodies against CYLD (1:1000, Cell Signaling Technology, USA), phospho-IKKβ (1:1000, Cell Signaling Technology), total IKKβ (1:1000, Cell Signaling Technology), total IκBα (1:1000, Cell Signaling Technology), RelA (1:1000, Cell Signaling Technology), Flag (1:1000; ProteinTech group, USA), proliferating cell nuclear antigen (PCNA) (1:2000, Biosynthesis, China) and GAPDH (1:3000, Biosynthesis). After incubation with HRP-linked secondary antibodies, the bands were visualized by western chemiluminscent HRP Substrate Kit (PPLYGEN, Beijing, China).

Protein from tissues was extracted with Pro-Orep (iNtRON Bio, Seongnam, Korea) according to the manufacturer’s instructions. Western blot analysis was performed with protein lysates using antibodies to sterol regulatory element-binding protein 1 (SREBP1; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and phospho-NF-κB, total NF-κB, phospho-IκBα, total IκBα, fatty acid synthase (FAS), and GAPDH (all from Cell Signaling Technologies, Danvers, MA, USA).

Antibodies against phospho‐Akt Ser⁴⁷³ (#9271), phospho‐Akt Thr³⁰⁸ (#9275), phospho‐TBC1D1 Thr⁵⁹⁰ (#6927), phospho‐JNK Thr¹⁸³/Tyr¹⁸⁵ (#9251), phospho‐p38 MAPK Thr¹⁸⁰/Tyr¹⁸² (#9216), phospho‐ERK1/2 Thr²⁰²/Tyr²⁰⁴ (#9101), total Akt (#9272), total TBC1D1 (#4629S), total‐JNK (#9252), total‐p38 MAPK (#9212), total‐ERK1/2 (#9102), total IκBα (#9242), and total‐acetyl CoA carboxylase (ACC, #3662) were from Cell Signaling Technology (Beverly, MA). Antibodies against phospho‐AS160 Thr⁶⁴² (#07‐802), phospho‐ACC Ser⁷⁹ (#07‐303), phospho‐TBC1D1 Ser²³⁷ (#07‐2268), and total AS160 (#07‐741) were from Millipore (Temecula, CA). Anti‐phospho‐AS160 Ser⁵⁸⁸ (#3028P2) was from Symansis Limited (Timaru, New Zealand). Anti‐GLUT4 antibody (#4670‐1704) was from Bio‐Rad AbD Serotec (Oxford, UK). Anti‐SPT2 antibody (ab23696) was from Abcam (Cambridge, MA). Horseradish peroxidase (HRP)‐conjugated anti‐rabbit IgG was from Biosource International (Camarillo, CA). HRP‐conjugated anti‐sheep IgG was from Millipore. HRP‐conjugated anti‐mouse IgG was from Santa Cruz Biotechnology. Enhanced chemiluminescence reagents (ECL, ECL plus, and ECL Prime) were obtained from GE Healthcare Life Sciences (Uppsala, Sweden). All other reagents were obtained from Sigma‐Aldrich (St. Louis, MO).

Estrogen and rapamycin were purchased from Sigma-Aldrich. The NF-κB inhibitor, JSH-23 was purchased from CalBiochem. The PERK inhibitor, GSK797800 was obtained from Toronto Research Chemicals. The STAT3 inhibitor, Stattic was purchased from Tocris. For western blotting, antibodies against NF-κB, phosphorylated-NF-κB, phosphorylated-IκBα, total-IκBα, phosphorylated-eIF2α, total-eIF2α, PERK, and PARP were purchased from Cell Signaling Technology. NF-κB, C/EBPβ, PERK and scrambled siRNAs were obtained from GE Dharmacon.

Procyanidin was purchased from Aladdin Co. Ltd. (Shanghai, China). Phorbolmyristate acetate (PMA), lipopolysaccharide (LPS), and ATP were purchased from Sigma-Aldrich (St. Louis, MO, USA). ATP was dissolved in ddH₂O (500 mM) and 0.1 M NaOH was used to adjust the pH value to 7.4 as stock solution. DSS was bought from MP Biomedical (Aurora, OH, USA). RPMI-1640 and fetal bowel serum were purchased from Life Technology (Carlsbad, CA, USA). Antibody for F4/80 was purchased from eBioscience (San Diego, CA, USA). Antibodies for NLRP3, phospho-p65, total p65, phospho-IKKα/β, total IKKα, anti-total IKKβ, phospho-IκBα, total IκBα and caspase-1 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody for apoptosis-associated speck-like protein containing a CARD (ASC) was purchased from Santa Cruz (Santa Cruz, CA, USA). Antibody for MMP9 was purchased from R&D System (Minneapolis, MN, USA). ELISA kits for human IL-1β were purchased from Dakewe Biotech Co. Ltd. (Beijing, China). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Antibodies against total ERK1/2 (#9102), phospho-ERK1/2 (Thr202/Tyr204; #9101), total IκBα (#9242), total JNK1/2 (#9252), phospho-p38MAPK (Thr180-Tyr182; #9211), total MK2 (#3042), phospho-MK2 (Thr334; #3042), total MSK1 (#3489), phospho-MSK1 (Thr581; #9595), phospho-c-Jun (Ser73; #9164) and phospho-IKKα/β (Ser176/180; #2697) were purchased from Cell Signaling Technology. Antibodies to Phospho-CREB (Ser133, #06-519) was from Millipore, anti-p38α (#sc-535) and anti-DUSP1 (#sc-373841) were from Santa Cruz, anti-active phospho-JNK1/2 (Thr183-Tyr185; #MAB1205) from R&D System, and anti-α-Tubulin (#T9026) from Sigma. Secondary antibodies from Invitrogen (Waltham, Massachusetts, United States) included Alexa Fluor 680 donkey α-sheep IgG (H + L) (#A21102), Alexa Fluor 680 goat α-rabbit IgG (H + L) (#A21109), Alexa Fluor 700 goat α-mouse IgG (H + L) (#A21036).
Kinase inhibitors SB203580 (inhibits p38α/p38β (Kuma et al., 2005 (link))) was purchased from Selleckchem, and SB747651A (inhibits MSK1, (Naqvi et al., 2012 (link))) from Axon Medchem. PD184352 (MKK1 inhibitor, (Kuma et al., 2005 (link))) and BIRB0796 (p38α/p38β/p38γ and p38δ inhibitor, (Kuma et al., 2005 (link))) were from the Division of Signal Transduction Therapy (DSTT); University of Dundee (Dundee, UK). JNK-IN-8 (JNK inhibitor (Zhang et al., 2012 (link))) was from Calbiochem.