Ab23426

For Western blot analysis, the treated cells were washed twice with ice-cold PBS and incubated in a cell lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing 1% phenylmethylsulfonyl fluoride (PMSF), a protease inhibitor, for 15 min at 4°C. Standard sodium dodecyl sulfate (SDS) sample buffer was added to the protein products, which were boiled for 10 min and centrifuged at 12,000×g for 10 min at 4°C. The sample proteins were then separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, Massachusetts, USA), which was blocked using 5% skim milk for 2 h at room temperature, and incubated with the specific antibodies against Notch3 and NICD3 (ab23426; Abcam, Cambridge, UK, 1: 5000) and anti-Bcl-2 and anti-Bax antibodies (YM3041 and YT0459 respectively; Immunoway, USA, 1: 1000) for one night. Mouse antibodies against β-actin were purchased from ZSGB-BIO (Beijing, China). Horseradish peroxidase (HRP)-conjugated IgG was used as the secondary antibody. Western blot analysis of β-actin on the same membrane served as the loading control. The protein bands were densitometrically analyzed using ImageJ software. All experiments were performed in triplicate.

The protein of treated ATDC5 cells was lysed using lysis buffer (Promega) supplemented with protease inhibitors (Roche, Mannheim, Germany). Each protein sample was quantified using the BCA™ Protein Assay Kit (Pierce, Appleton, WI, USA). Then, proteins were separated by SDS-PAGE and were blotted onto polyvinylidene difluoride membranes, followed by blocking with 5% non-fat milk. The blocked membranes were respectively incubated with anti-Bcl-2-associated X protein (Bax, ab182733), anti-B cell lymphoma-2 (Bcl-2, ab196495), anti-pro caspase-3 (ab90437), anti-cleaved caspase-3 (ab49822), anti-p65 (ab16502), anti-phosphorylated p65 (p-p65, ab86299), anti-inhibitor of nuclear factor κB α (IκBα, ab32518), anti-phosphorylated IκBα (p-IκBα, ab133462), anti-Notch1 (ab52627), anti-Notch2 (ab137665), anti-Notch3 (ab23426), anti-GAPDH (ab181603) (all Abcam, Cambridge, UK), anti-pro caspase-9 (9508), anti-cleaved caspase-9 (9509) (both Cell Signaling Technology, Beverly, MA, USA), or anti-PDCD4 (sc-376430; Santa Cruz, Santa Cruz, CA, USA) at 4 °C overnight. Then, after rinsing thrice, membranes were incubated with secondary antibodies marked by horseradish peroxidase for 1 hr at room temperature. After rinsing again, the bands on the membranes were visualized by ECL detection systems (Sigma-Aldrich).

Niclosamide was obtained from Sigma-Aldrich (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; also known as thiazolyl blue and methylthiazolyldiphenyl tetrazolium bromide) was purchased from Sigma-Aldrich. The Annexin V-FITC Apoptosis Detection kit was purchased from Vazyme Biotech (Nanjing, China). Dimethyl sulfoxide (DMSO) was obtained from BioSharp (Hefei, China). The primary antibodies, rabbit polyclonal anti-Notch1 (ab27526), rabbit polyclonal anti-Notch2 (ab8926), rabbit polyclonal to anti-Notch3 (ab23426) and rabbit polyclonal anti-Hey1 (ab22614) were purchased from Abcam (Cambridge, UK). Mouse anti-β-actin monoclonal antibody (TA-09) was purchased from ZSGB-BIO (Beijing, China). The secondary antibodies, peroxidase-conjugated AffiniPure goat anti-rabbit IgG (ZB-2301) and peroxidase-conjugated AffiniPure goat anti-mouse IgG (ZB-2305), were purchased from ZSGB-BIO.