Ab59195

After lysing in RIPA buffer, the collected total protein was separated on 12% SDS-PAGE and shifted onto PVDF membranes. 5% nonfat milk powder was added for blocking membranes. Next, the membrane was probed all night at 4 ​°C with primary antibodies against loading control GAPDH (ab8245, 1/1000; Abcam, Cambridge, MA, USA), TRAP (ab65854, 1/1000; Abcam), NFATc1 (ab175134, 1/1000; Abcam), TRAF3 (ab36988, 1/1000; Abcam), TRAF1 (ab129279, 1/1000; Abcam), TRADD (ab110644, 1/1000; Abcam), TRAF2 (ab230795, 1/1000; Abcam), ADAM17 (ab39162, 1/1000; Abcam), TNFRII (ab8161, 1/1000; Abcam), TNFRSF1A (ab19139, 1/1000; Abcam), p-IKB (ab133462, 1/1000; Abcam), IKB (ab32518, 1/1000; Abcam), TNFRSF11 (NB100-56508, 1/1000; Novus Biologicals, Littleton, CO, USA), p-IKKα (ab38515, 1/1000; Abcam), IKKα (ab32041, 1/1000; Abcam), p-IKKβ (ab59195, 1/1000; Abcam), IKKβ (ab124957, 1/1000; Abcam), p-Erk (ab214036, 1/1000; Abcam), Erk (ab184699, 1/10000; Abcam), p-MEK (ab96379, 1/1000; Abcam), MEK (ab32091, 1/1000; Abcam). On the following day, the HRP-secondary antibodies were added for 2 ​h at room temperature. The protein density was examined by the ECL luminous liquid (Pierce, Rockford, IL, USA). The assay was taken in triplicate.

The protein levels of p65, IKK, p‐IKKβ, IκBα, and p‐IκBα in laryngeal cancer cells were detected by performing immunoblotting. We lysed cultured or transfected cells in RIPA buffer with 1% PMSF and loaded protein onto an SDS‐PAGE minigel and transferred them onto PVDF membrane. The blots were probed with the following antibodies: anti‐p65 (ab16502; Abcam, Cambridge, MA), anti‐IKK (ab178870; Abcam), anti‐p‐IKKβ (phospho Y199; ab59195; Abcam), anti‐IκBα (#9242; Cell Signaling Technology, Danvers, MA), anti‐p‐IκBα (Ser 32; sc‐7977; Santa Cruz, Dallas, TX), anti‐β‐actin (ab6276; Abcam), and anti‐Lamin B (ab133741; Abcam) at 4°C overnight, subsequently incubated with HRP‐conjugated secondary antibody (1:5000). ECL substrates were used to visualize signals (Millipore, MA). β‐actin was used as an endogenous protein for normalization.

Glycyrrhizic acid (DG0006) was obtained from Chengdu DeSiTe Biological Technology Co., Ltd (China). Zinc sulfate (A602906) and galactose (A600215) were purchased from Sangon Biotech (China). PEI (E8420), Hemotoxylin and Eosin staining kit (G1120) and RIPA lysis buffer (R0010) were purchased from Solarbio Life Science (China). DSPE-PEG 2000 was produced by Xi'an Ruixi Biological Technology Co., Ltd. (China). Penicillin–streptomycin, fetal bovine serum (FBS) and Dulbecco's modified Eagle’s medium (DMEM) were produced by Gibco (USA). Free fatty acids (1170460001), palmitic acid (P0500) and Oil Red O (O0625) were obtained from Sigma (USA). p-IKKβ (Abcam, ab59195, 1:1000), IKKβ (Abcam, ab124957, 1:1000), p-p65 (ab86299), p65 (ab16502), TNF-α (ab183218), IL-6 (ab259341), ABCA1 (ab66217), CPT1 (ab234111) primary antibodies and horseradish peroxidase (HRP)-labeled secondary antibodies were obtained from Abcam (USA).

Frozen placenta tissues and collected cells were taken out from liquid nitrogen and lysed with RIPA lysis and extraction buffer (89901, Thermo Fisher Scientific, USA). The manufacturer's instruction was strictly followed. The protein samples were collected from the supernatants after centrifugation. Protein samples were then loaded into SDS-PAGE gels to run for electrophoresis, followed by PVDF membrane transfer and skim milk blocking processes. Postblocking membranes were incubated at room temperature with primary antibodies against PPARα (ab24509, Abcam, USA), CD36 (ab133625, Abcam, USA), acyl-CoA oxidase (ACO) (ab248375, Abcam, USA), uncoupling protein 2 (UCP2) (ab97931, Abcam, USA), NF-κB (ab131546, Abcam, USA), IKKβ (ab124957, Abcam, USA), p-IKKβ (ab59195, Abcam, USA), IκBα (ab7217, Abcam, USA), and p-IκBα (ab133462, Abcam, USA). The primary antibodies against GAPDH (ab181602, Abcam, USA) and histone H3 (ab1791, Abcam, USA) were separately used as loading controls in total cell proteins and nucleus proteins. An HRP-conjugated goat anti-rabbit secondary antibody (ab6721, Abcam, USA) was applied to the membranes after the removal of primary antibodies, followed by an incubation of 1 hour at room temperature. SuperSignal West Pico PLUS Chemiluminescent Substrate (34580, Thermo Fisher Scientific, USA) was used during the grey analysis by using an imaging system.

Total protein was extracted from cultured cells and liver tissues by using ice cold RIPA lysis buffer. The protein concentration was determined by BCA protein assay kit (Solarbio, Beijing, China). 20 μg of protein was ran on a 10% SDS-PAGE gel and transferred to a PVDF (Millipore, USA) membrane. GAPDH was used as a control and non-specific bindings were blocked by 5% BSA for 40 min at room temperature. Incubates the specific primary antibodies against p-IKKβ (Abcam, ab59195, 1:1000), IKKβ (Abcam, ab124957, 1:1000), p-p65 (Abcam, ab86299, 1:1000 dilution), p65 (Abcam, ab16502, 1:1000 dilution), TNF-α (Abcam, ab183218, 1:1000 dilution), IL-6 (Abcam, ab259341, 1:1000 dilution), ABCA1 (Abcam, ab66217, 1:1000), CPT1 (Abcam, ab234111, 1:1000) at 4 ℃ overnight. After extensively washing with 100 µM PBST, peroxidase-conjugated secondary antibody (Abcam, ab205718, 1:5000) was applied to the membranes and incubated for 2 h at room temperature. Protein bands were detected by using ECL substrates (Thermal Fisher, Rockford, USA) and visualized under a ChemiDoc XRS + imager (Bio-Rad, Hercules, USA).