Anti pcna antibody

We used a lysis buffer to isolate total proteins in MG-63 cells (Beyotime Biotechnology, Shanghai, China). We used the BSA assay to test the protein concentration (Amresco, OH, USA). Then, in equal concentration, SDS-PAGE was carried out with proteins as described earlier in literature 14. Abcam Biotechnology (MA, USA) supplied anti-PDZD2 antibody (ab133324, 1:1000), anti-ALCAM antibody (ab109215, 1:1000), and anti-GAPDH antibody (ab8245, 1:10000). Cell Signaling Technology (MA, USA) supplied anti-E-cadherin antibody (#3195, 1:1000), anti-vimentin antibody (#35741, 1:10000), and anti-PCNA antibody (#13110, 1:1000). The enhanced chemiluminescence technique was used to visualize the bands of proteins.

After fixation and routine dehydration, all tumor samples were embedded in paraffin and cut into 2-μm thick sections. The xenografted specimens for histological analysis were stained with hematoxylin and eosin (HE) in order to observe the general tissue morphology under the DM4000B microscope (Leica, Germany). For immunohistochemistry, sections were deparaffinized using xylenes for 10 min each and hydrated using a graded alcohol series (100 to 75%) for 5 min each. Antigen retrieval was performed by heating the sections in citrate buffer for 2 min in a pressure cooker. The endogenous peroxidase activity was inactivated using 0.3% hydrogen peroxide for 10 min at RT in the dark. Afterwards, sections were incubated with a 1:8000 dilution of anti-PCNA antibody and 1:800 dilution of anti-P62 antibody (Cell Signaling Technology, USA) overnight in a moist chamber at 4 °C. The next day, the sections were washed and incubated with an HRP-conjugated rabbit secondary antibody (Cell Signaling Technology, USA) for 30 min at RT. The PCNA and p62 signals were detected by using DAB substrate (brown). All tumor sections were counterstained with haematoxylin for 1 min, dehydrated, dried, and mounted using Permount TM Mounting Medium. The images were captured using the LEICA DM4000 B LED microscope (Leica, Germany).

Cell lysate combinations of RIPA with PIC2 and PPI were used to form the lysis buffer, and a reaction time of 30 min in an ice bottle was allocated. The protein concentration in each cell lysate was then measured using a commercial BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). In the Western blot analysis, an SDS-polyacrylamide gel electrophoresis system was used. An anti-GAPDH antibody (Cell Signaling, Danvers, MA, USA), anti-fibronectin antibody (Abcam, Cambridge, England, UK), anti-vimentin antibody (cell signaling), anti-E-cadherin antibody (BD Biosciences, Franklin Lakes, NJ, USA), anti-ZO-1 antibody (BD Biosciences), anti-PCNA antibody (Cell Signaling), anti-protein kinase B (AKT) antibody (Cell Signaling), anti-phospho-AKT (P-AKT) antibody (Cell Signaling), anti-α7-nAChR antibody (Abcam), and anti-SNCG antibody (Cell Signaling) were used for probing.

Freshly dissected liver samples were fixed in 10% formalin for 24 h and embedded in paraffin. For hematoxylin and eosin (HE) staining, 4-µm tissue sections were stained with HE. For immunohistochemistry, cleaved caspase-3, γ-H2AX, 4-HNE, PCNA and Ki-67 were labeled in paraffin-embedded liver sections using an anti-cleaved caspase-3 antibody, anti-PCNA antibody, anti-Ki-67 antibody (Cell Signaling Technology), and anti-4-HNE antibody (Abcam), respectively. The detection of immunolabeled proteins was performed using an avidin–biotin complex with the Vectastain ABC Kit (Vector Laboratories). Four visual fields of a magnified image (× 20 or × 40) were randomly selected for each immune-stained section, and the positive cell ratio was calculated.

Anti-MPG antibody (Proteintech, Chicago, IL, USA), anti-XRCC1 and anti-APEX1 antibodies (Abcam, Cambridge, England), anti-POL β antibody (Trevigen, Gaithersburg, MD, USA), anti-β-Actin antibody (Sigma-Aldrich, St Louis, MO, USA) and anti-PCNA antibody (Cell Signaling Technology, Danvers, MA, USA) were used in the study.

Cells were lysed in RIPA lysis buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF). Total protein lysates (25 μg) were fractionated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to PVDF membranes, and blocked at room temperature for 1 h with 5% nonfat dry milk and followed by overnight incubation with the anti-CPNE3 antibody (1:500, Abcam, Ab236606), anti-PCNA antibody (1:1000; Cell Signaling Technology, #13110), anti-ki67 antibody (1:500, Abcam, Ab16667), anti-XIAP antibody (1:1000, Abcam, Ab28151), anti-Bim antibody (1:1000, Abcam, Ab7888), anti-p-P13K antibody (1:1000, Abcam, Ab182651), anti-P13K antibody (1:1000, Abcam, Ab133595), anti-AKT antibody (1:1000, Cell Signaling Technology, #9272), anti-p-AKT antibody (1:2000, Cell Signaling Technology, #4060), anti-GAPDH antibody (1:2000, Cell Signaling Technology, #5174). An HRP-conjugated secondary antibody (1:1000, Beyotime) was applied for 1h at room temperature. The intensity for each protein band was corrected by the intensity of the GAPDH band and was normalized to facilitate comparisons.