Hrp linked goat anti mouse igg

The total protein extraction was performed from HeLa cells only, HeLa cells treated with EGCG (25 μM), cisplatin (250 nM), and EGCG co-treated with cisplatin for 24 h. Sample proteins were separated by sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and subsequently transferred to a nitrocellulose membrane. Then, blots on the membrane were incubated with blocking solution (5% non-fat dry milk). Primary antibody was diluted (1:1000) in the same buffer containing 0.05% Tween-20. Afterwards, the blots were incubated overnight with primary antibody, anti-NF-κB p65, anti-COX-2, anti-HO-1, anti-p-mTOR, anti-p-p70S6K1, antip4E-BP1, anti-p-Akt (Abcam, Cambridge, UK), and anti-Nrf2 (Santa Cruz, CA, USA) at 40°C and on the following day, with secondary antibody (HRP-linked goat anti-mouse IgG, Abcam, Cambridge, UK) for 1 h at room temperature. Blots were then incubated with diaminobenzidine and H₂O₂ as substrates for visualization of specific binding. Protein loading was controlled using a monoclonal mouse antibody against β-actin (A5316; Sigma). Blotting was performed at least three times to confirm data reproducibility. Immunoreactive protein bands were quantified densitometrically using ImageJ analysis system (NIH, Bethesda, USA). Results were normalized to the β-actin expression in each group as percent of control. Blots were performed three times.

Dulbecco's modified Eagle's medium (DMEM/F12), foetal bovine serum (FBS) and collagenase II were obtained from HyClone. Trypsin was provided by Beyotime. All other cell culture reagents were purchased from Sigma Chemicals unless otherwise specified. Primary antibodies against KDM3A, ETS‐1, Bax, Bcl‐2, IL‐6, TNF‐α, GAPDH, ANP and BNP were purchased from Abcam. The secondary antibodies, either HRP‐linked goat anti‐mouse IgG or goat anti‐rabbit IgG, were also obtained from Abcam. The miR‐22‐3p mimic and its scrambled oligonucleotides (miRNA‐NC) were synthesized by GenePharma Co., Ltd.

The HeLa cell extracts were obtained with PBS-1% TritonX-100 (Sigma) containing a lysis buffer, and 20 µg of protein preparation was loaded onto 10% acrylamide gels in each lane. Proteins in samples were fractionated by electrophoresis and migrated on 10% SDS-PAGE, then transferred to a nitrocellulose membrane. Blocking was carried out using 5% dry milk for 2 h. Then, membranes were incubated overnight with diluted (1:1000) primary antibody, anti-Nrf2, anti-Bcl-2, anti-Bax, and anti-NF-κB p65 (Santa Cruz, CA, USA) at 40 °C. Antibody labeling was detected by incubation with a secondary antibody (HRP-linked goat antimouse IgG, Abcam, Cambridge, UK). A monoclonal mouse β-actin antibody (A5316, Sigma) was used to control the loading of protein. Band quantification analysis was done using ImageJ software (NIH, Bethesda, USA). Standardization of results was achieved using the β-actin expression as a percentage of control in each group. Blots were performed in triplicate to confirm the reproducibility of data.