Hrp linked secondary antibody

SA, APZ, and imipramine (IMP) were purchased from Sigma–Aldrich (St Louis, MO, USA). Acetonitrile and methanol (UPLC grade) were obtained from Panreac Chemicals (Barcelona, Spain), while potassium dihydrogen phosphate was procured from Winlab Ltd. (Maidenhead, Berkshire, UK). Milli-Q water was prepared in the laboratory by a purification system (Millipore Corp., Billerica, MA, USA). Anti-CYP3A2 (LS-C36108), CYP2D6 (PA1397) and anti β-Actin (sc-47778) antibodies were purchased from Lifespan Biosciences, Inc. (Seattle, WA, USA), Boster Bio, (Pleasanton, CA, USA) and Santa Cruz Biotechnology, Inc. (Dallas, Texas U.S.A), respectively. HRP-linked secondary antibodies were procured from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), and Luminata Forte Western HRP substrate was obtained from Millipore (Billerica, MA). All other chemical reagents used were of an analytical grade.

Cells after treatment, were extracted with RIPA lysis buffer (Biyuntian, Hangzhou, China). Protein lysates were then separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with the primary antibodies: P21 (1:1,000), Ki-67 (1:1,000), PCNA (1:1,000), MMP-2 (1:1,000), MMP-9 (1:2,000), FOXM1 (1:1,000) and β-actin (1:1,000) (all from Abcam, Cambridge, MA, USA) overnight at 4°C. Then, the membranes were incubated in HRP-linked secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 2 h. Western blotting signals were detected using the ECL Plus kit (Biyuntian). Each experiment was repeated 3 times, independently.

Laemmli sample buffer was added to the cell lysate samples and these were run on 8–10% SDS polyacrylamide gels and blotted onto nitrocellulose membranes. Membranes were blocked in phosphate buffered saline containing 0,1% Tween- 20% and 5% dry milk. The first antibodies were diluted in phosphate buffered saline containing 0,1% Tween- 20% and 5% bovine serum albumin and used either for 2 h at room temperature or overnight at 4 °C. After several washing steps in PBS-Tween-20, membranes were incubated with HRP-linked secondary antibodies (Santa Cruz Biotechnology, Inc., USA) diluted in blocking buffer. Antibody binding was detected using enhanced chemiluminescence and Fuji Super RX medical X-ray films.
For the detection of collagen IV NC1 monomers and dimers, cells or small tissue pieces were harvested in hypotonic lysis buffer (10 mM CaCl₂, 50 mM Hepes, pH 7.4) completed with 0.1 mM benzamidine hydrochloride, 25 mM 6-aminocaproic acid, 1 mM Phenylmethylsulfonyl fluoride and 0.5 mg/ml type I collagenase at 37 °C for 12–18 h. For the lysis of hypoxia/anoxia treated and control cells, the lysis buffer was supplemented also with 150 µM phloroglucinol to inhibit PXDN activity during the lysis process.