Pnf κb

Full-length coding sequences of PTPN14 and SOCS7 (NCBI accession number: NM_008976, NM_138657) were inserted into pCMV-HA, pCMV-Myc, or pGEX-4T-1 vectors. Primers are as follows: HA-PTPN14, F-AACTGTCGACCATGCCTTTCGGC, R-TATAAGCGGCCGCATTGTGTGTAT; Myc-SOCS7, F-AATCGAATTCCGATGCAGGGGG, R-CCCCAGGGCTACAGTCGACTTAA; GST-PTPN14, F-AATCGTCGACCCATGCCTTTCGG, R-TATAAGCGGCCGCATTGTGTGTAT. The WT Ubiquitin and other mutants were purchased from Addgene. The pGL6-TA and pNF-κB used in the measurement of NF-κB activity were purchased from Beyotime (Jiangsu, China).

RIPA buffer (Beyotime, China) was used to lyse cells to obtain total protein, and the protein concentration was determined by BCA method. The same amount of samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein was transferred to the PVDF membrane (Millipore, Burlington, United States). After blocking with 5% skim milk powder (Beyotime, China) for 2 h at room temperature, the membrane and antibody were incubated overnight at 4°C. The primary antibodies were as follows: AMPK, NF-κB, p-NF-κB, MMP9, MMP2, TIMP-2, E-cadherin, Vimentin and β-actin (Beyotime, China). Then incubated with HRP-labeled Goat Anti-Rabbit IgG (H + L) (Beyotime, China) at room temperature for 2 h. Protein bands were detected by enhanced chemiluminescence (Tanon chemiluminescent gel imaging system, Tanon-5200Multi, China).

Carrageenan was acquired from Sigma-Aldrich (Shanghai, China). PI, FBS, and PBS were acquired from Servicebio (Wuhan, China). ALT, AST, ALP, TBIL, and DBIL kits were obtained from Biobase (Jinan, China). NF-κB, P-NF-κB, β-actin, and IκB antibodies were obtained from the Beyotime Institute of Biotechnology (Shanghai, China). RIP1, RIP3, and IKKα antibodies were obtained from Proteintech (Wuhan, China). 3-MA and Nec-1 were acquired from MedChem Express (Shanghai, China). LDH cytotoxicity assay kit and Z-VAD-FMK were obtained from the Beyotime Institute of Biotechnology (Shanghai, China). DPH was acquired from Aladdin (Shanghai, China). DMEM, DMSO, MTT, FDA, and PVDF membranes were obtained from Solarbio (Beijing, China). Other chemicals were of analytical grade and commercially available.