Anti mouse antibody

Uninfected normal and E6E7 HPV16 positive 3D cultures were processed by both IF and biochemical analysis.
In vitro cultures slides were ON incubated at 4 °C with the following primary antibodies: mouse anti-γH2A.X (clone JBW301, working dilution 1:400, EMD Millipore Corp., USA, distributed by Merck, Darmstadt, Germany) and rabbit polyclonal anti-53BP1 (ab36823, working dilution 1:1000, abcam).
For antigens retrieval, all sections were incubated in 1 mM EDTA pH 8 for 50 min at 95 °C. γH2A.X and 53BP1 incubations were respectively followed by the addition of Alexa Fluor 568 and 488 dyes conjugated specific secondary antibodies, according to the manufacturer’s instructions, and TSA signal development (Fig. 3A-C, C’, C″).
Protein extracts were then obtained, run, transferred and detected as above described. Membranes were probed with the following primary antibodies: anti-γH2A.X (working dilution 1:1000), anti-53BP1 (working dilution 1:1000) and anti-tubulin as internal control (Fig. 3B). A peroxidase-coupled goat anti-rabbit (working dilution 1:2000, abcam) or anti-mouse antibody (working dilution 1:2000, abcam) incubation was made. Antigens were then revealed as above described.

Western blot analysis was performed in lung cancer cells with different treatments. After aspirin administration, total proteins were obtained from the different cell groups and the proteins in equal measure from each group were examined. The primary antibodies used in this study, anti‐TAZ or anti‐β‐actin, were purchased from Cell Signaling Technology (USA) or Santa Cruz Biotechnology (USA). The secondary antibodies including goat anti‐rabbit (Abcam, USA) or anti‐mouse antibody (Abcam) were incubated with the blots and visualized through an enhanced ECL chemiluminescent substrate kit (Roche, Switzerland).

Proteins were isolated from whole tissue using radio immunoprecipitation assay (RIPA) buffer as described earlier [22 (link)]. Protein concentration was determined with Bradford reagent (Bio-Rad, USA). 30 μg/µl of protein was loaded on 12% SDS-PAGE gel and electrophoresis was done. Later, proteins were transferred to a nitro cellulose membrane. Nonspecific binding sites were blocked by placing the membranes in 5% BSA in TBS for 1 h. Primary antibody was added and incubated at 4°C for overnight with mild shaking. Primary antibodies in this study were used in following dilutions, anti Hey2 antibody (1:500), anti Dll4 antibody (1:1000), anti Ephrin B2 antibody (1:500), anti COUP-TFII antibody (1:800), anti Endoglin antibody (1:1000), anti KLF2 antibody (1:1000), anti GGTP antibody (1:1000) and anti GLUT1 antibody (1:1000). Subsequently, membranes were probed with horse radish peroxidase (HRP) conjugated secondary antibodies (Anti-rabbit (1:5000), Abcam, U.K and Anti-mouse antibody (1:5000), Abcam, U.K). Finally, the bands were detected using enhanced chemiluminescence (ECL) (Bio-Rad) and the band was transferred to Kodak X-ray film.

The BrdU assay was performed according to a previous study [23 (link)]. U251 and U87 cells were seeded at a density of 1 × 10⁴ cells/well in 96-well plates. After cells reached 80% confluence, the cells were labeled with BrdU (Cat#: ab126556, Abcam, Cambridge, UK) for 12 h. After washing twice with PBS, the cells were permeabilized and incubated with BrdU antibody for 2 h at room temperature. Then, the cells were incubated with anti-mouse antibody (Abcam, Cambridge, UK) for 1 h and subjected to a multimode-plate-reader at OD 450 nm (Thermo Fisher Scientific).