Proliferating cell nuclear antigen (pcna)

Protein was extracted from hUC-MSC samples or liver tissue using radioimmunoprecipitation assay (RIPA) lysis buffer. The primary antibodies were insulin receptor substrate 1 (IRS-1), phosphoinositide 3-kinase (PI3K, 1:1200, Immunoway Biotechnology), protein kinase B (AKT, 1:1200, Immunoway Biotechnology), phosphoinositide-dependent protein kinase 1(PDK1, 1:1200, Immunoway Biotechnology), glucose transporter type 4 (GLUT4, 1:1200, Immunoway Biotechnology), B-cell lymphoma-2-associated X (BAX, 1:1200, Immunoway Biotechnology), proliferating cell nuclear antigen (PCNA, 1:1200, Immunoway Biotechnology), B-cell lymphoma-2 (BCL-2), cell cycle-associated proteins (Ki-67, cyclin A, cyclin E, 1:1200, Immunoway Biotechnology), A 160 kDa substrate of the Akt Ser/Thr kinase (AS160, 1:1200, Immunoway Biotechnology), cysteine protease protein (Caspase3, 1:1200, Immunoway Biotechnology) glyceraldehyde phosphate dehydrogenase (GAPDH, 1:3000), and β-actin (1:3000). The secondary antibodies conjugated to horseradish peroxidase (1:3000, Immunoway Biotechnology). The normalization of other proteins was performed using β-actin and GAPDH western blot assays [19 (link)].

The cells were washed with PBS before being lysed in lysis buffer that can extract both cytoplasm proteins and nucleoproteins. The sample was stored at −80°C. Samples containing an equal amount of proteins were mixed with 5x SDS loading buffer and electrophoresed on a 10% SDS-PAGE gel. The proteins were then transferred onto PVDF membrane (Millipore). The membranes were blocked and probed with antibodies anti-NFAT2 (CST), TRAF6 (Immunoway), p-NF-κBp65 (Immunoway), PCNA (Immunoway), p65 (Immunoway), and β-actin (Proteintech group). The membranes were incubated with specific primary antibodies overnight at 4°C at a 1 : 1000 dilution. Subsequently, the membranes were washed with TBS/T for 15 min, three times, and incubated for 1 h with HRP-conjugated secondary antibodies (Abbkine). The protein was visualized with chemiluminescence, and a densitometric scanner was used to determine the density of the band. All experiments were repeated at least three times independently.