Pnf kb p65

Cultured cells were lysed in a radio-immunoprecipitation assay buffer and proteins were fractionated by 10-15% SDS gels and electro-transferred to polyvinylidene difluoride transfer membranes (Millipore, Billerica, MA, USA). The membrane was incubated overnight with primary antibodies and then with secondary antibodies conjugated with horseradish peroxidase (Cell Signaling, Danvers, MA, USA). Antibodies against c-Myc, Oct4, Sox2, Klf4, Lrp5, Runx2, Snail, TGFβ, Eno1, Eef2, vinculin, β-catenin, p-Akt, Akt, p-NFkB p65, NFkB p65, p-ERK, ERK (Cell Signaling), MMP9, NFATc1, cathepsin K (Santa Cruz, Dallas, TX, USA), TRAIL (Novus, Centennial, CO, USA), p53 (Invitrogen, Carlsbad, CA, USA), Hsp90ab1 (Abcam, Cambridge, UK), Kdm3a (Thermo Fisher Scientific) and β-actin as a control (Sigma) were employed. The level of proteins was determined using a SuperSignal west femto maximum sensitivity substrate (Thermo Fisher Scientific), and a luminescent image analyzer (LAS-3000, Fuji Film, Tokyo, Japan) was used to quantify signal intensities.

Western blotting was performed to evaluate the protein expression levels of DEGs. The liver tissues were homogenized using a liquid nitrogen pre-cooled high-speed tissue homogenizer (Gering Scientific Instruments Ltd, Beijing, China) and lysed using 10 μM phenylmethanesulfonyl fluoride (PMSF, Beyotime Institute of Biotechnology, Jiangsu, China) and a 1% protease inhibitor cocktail (104 mM AEBSF, 80 μM Aprotinin, 4 mM Bestatin, 1.4 mM E-64, 2 mM Leupeptin, and 1.5 mM Pepstatin A; Sigma). The protein contents were analyzed with the BCA Protein Assay Kit (Vazyme Biotech Co., Ltd, Nanjing, China). Standard Western blotting was performed, and the blots were visualized via a chemiluminescent system using ImageQuant LAS 500 imaging instruments (GE Healthcare Life Sciences, Shanghai, China) and quantified using the Image J analyzer software. Antibodies against ERK 1/2, p-ERK1/2, p-NF-kB p65, MAPK p38, p-MAPK p38, JNK, and p-JNK were purchased from Cell Signaling Technology (Danver, MA, USA). Antibodies against TLR4, NLRP3, IL-1b, MYD88, and NF-kB p65 were obtained from Abcam (Cambridge, MA, USA).

Total protein was extracted using RIPA lysis buffer (50mM Tris-HCl, 150nM NaCl, 1% IGE-PAL, 0.5% sodium deoxycholate, 0.1% SDS, protease, and phosphatase inhibitor cocktail (Roche Applied Science, Bavaria, Germany)). A total of 25 µg of protein was loaded into each well of SDS-PAGE gels with 4% stacking and 10% running buffer (Bio-Rad Laboratories, Inc.). The proteins were blotted onto the PVDF membrane (Bio-Rad Laboratories, Inc.) and blocked with blocking buffer (TBS buffer contained 0.5% Tween-20 and 4% skim milk). The membrane was then incubated with primary mouse monoclonal antibody against HMGA2 (Abcam, ab97276), NF-kB-P65 (Cell signaling technology, 8242T), p-NF-kB-P65 (Cell signaling technology, 3031S), STAT3 (Cell signaling technology, 9139S), p-STAT3 (Cell signaling technology, 9145S), and GAPDH (Santa Cruz Biotechnology, SC-32233) overnight at 4 °C on a shaker. Next, the membrane was incubated with secondary goat anti-mouse or mouse anti-rabbit antibodies conjugated with HRP (Dako Corporation) on a shaker for 1h at room temperature. The protein bands were developed with ECL reagent (Bio-Rad Laboratories, Inc.) and visualized on autoradiography films.