Pcna antibody

The antibodies anti-RECQ1 (ab151501, 1:200 dilution), anti-53BP1 (ab175933, 1:200 dilution), anti-RPA (ab2175, 1: 1000 dilution), anti-PARP1 (ab191217, 1:1000 dilution) and anti-BrdU (ab6326, 1:200), anti-RPA2-pS4/S8 (ab87277, 1:200 dilution) were purchased from Abcam. Antibodies anti-γH2AX (2577, 1:800 dilution), α-Tubulin (2144, 1:1000 dilution) and PCNA antibody (2586, 1:200 dilution) were purchased from Cell Signaling. Mouse anti-BrdU (347580, 1:40) was purchased from BD Biosciences. AF647 (A-21247, 1:1000) and AF488 (A-11001, 1:1000) were purchased from Thermo Fisher Scientific. BMN673 (S7048, Talazoparib) was purchased from Selleck Chemicals.

For the analysis of PCNA by fluorescence microscopy, cells were seeded onto the glass coverslips in 24-well plates. After synchronization, cells were fixed in 2% PFA for 20 min and were permeabilized in 1% triton X-100 in PBS 10 min at room temperature. Then, cells were incubated with PCNA antibody (Cell signaling) and visualized with Alexa 488-conjugated igG (Molecular Probes). Nucleus staining was performed with Hoechst 33342 (Molecular Probes). The immunofluorescence was visualized using a confocal microscopy (Carl Zeiss, LSM700).

Liver tissues extracts were prepared from liver cancer tissue specimens and the cell protein from HUVEC treated with the RIPA buffer. Then, an equal amount of protein (50 μg) was loaded onto 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed dried milk buffered for 2 hours at 25°C and incubated with primary antibodies against STAT3 rabbit mAb (dilution, 1 : 1,000; cat. no. 9959; Cell Signaling Technology, Inc., Danvers, MA, USA), p-STAT3 antibody (dilution, 1 : 1,000; cat. no. 9914; Cell Signaling Technology, Inc.), and PCNA antibody (dilution, 1 : 1,000; cat. no. 2586; Cell Signaling Technology, Inc.), overnight at 4°C. Next, the membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody for 2 hours at 25°C. The membranes were washed four times with tris-buffered saline with Tween-20 and detected by a fluorescence visible imaging system BIO-RAD imaging system (BIO-RAD, Hercules, CA, USA). The intensity of each band was evaluated by the densitometry test using Image Pro Plus 4.5 software (Media Cybernetics, Inc., Bethesda, MD, USA). The quantification was normalized to the comparable value of GAPDH expression.

Initially, sections were incubated with citrate buffer (pH 6.0) for antigen retrieval and then blocked in 5% NGS for 1 h and incubated at a dilution of 1:400 at 4 °C overnight with PCNA antibody (mouse monoclonal antibody, #2586, Cell Signaling Technology, Danvers, MA, USA). After washing with PBS, sections were incubated with Alexa Fluor 568 secondary antibody (1:500, Thermo Fisher Scientific, Waltham, MA, USA). Sections were examined using an Olympus FV1000 confocal microscope.

Cocaine hydrochloride, SU5416 (antagonist of VEGFR-2), and BD1047 (antagonist of sigma receptor) was obtained from Sigma Aldrich (St. Louis, MO). HIV-1 Tat 1–72 was purchased from University of Kentucky College of Medicine (Lexington, KY). U0126, phosphorylated ERK (Thr202/Tyr204) and PCNA antibody were purchased from Cell Signaling Technology (Beverly, MA). Glutathione, α-tocapherol and β-integrin antibody were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). L-ascorbate acid sodium salt was obtained from ACROS Organics (Belgium). ZO-1 antibody for immunocytofluorescence staining was purchased from Life Technologies (Carlsbad, CA). ZO-1 antibody for Western blot, Ras activation assay kit, In- vitro vascular permeability assay kit, and compartmental protein extraction kit were purchased from EMD Millipore (Billerica, MA).

Immunohistochemistry (IHC) staining was carried out on 4μm sections heated for 30 minutes at 60°C using the Bond III fully automated staining system with their Bond Polymer Refine detection system and associated reagents supplied by Leica Microsystems (Newcastle-Upon-Tyne, UK). Antigen retrieval and dilution was performed at 100°C for 30 min with MGMT antibody (Millipore, Thermo Fisher, UK) or PCNA antibody (Cell Signaling Technologies, 2586s) at a 1:100 dilution with Epitope Retrieval Solution 1(pH 6.0). Primary antibodies were applied to the section for 30 minutes. Slides were deparaffinized and pretreated with steam disodium ethylene diamine-tetraacetate at pH 8.0. A 30 minute incubation time with the primary antibody was performed. The primary antibody was substituted with mouse IgG1 at a dilution of 1:200 as a negative control. In accordance with previous reports, MGMT was considered positive when uniform MGMT staining was detected in cell nuclei [46 (link)].

The isolated liver tissues from the treated mice were fixed in 10% formalin for 24 h followed by processing and paraffin embedding. Sections (4μm) of paraffin-embedded tissues were stained with hematoxylin and eosin (H&E). Immunohistochemical (IHC) staining was performed using CD5L antibody (#50020-T24, Sino Biological, Beijing, China), PCNA antibody (#13110, Cell Signaling Technology, Beverly, MA, USA) and sheep anti-APAP polyclonal antibody (#0016-0104, Bio-Rad, Düsseldorf, Germany). Tissue sections stained with specific antibodies were evaluated by light-microscopic (Olympus IX51, Japan). Pathological images of tissue sections were analyzed by Image J (1.51j8).