Ab2618

After treatment, cells were washed twice with PBS and lysed on ice for 30 min with whole‐cell extract lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA). 20 µg total protein of each sample was resolved on the 4%–12% gradient SDS‐PAGE and transferred onto PVDF membrane (Bio‐Rad, USA) at 350 mA for 2 hr. For ATM detection, 3%–8% gradient SDS‐PAGE was used and PVDF membrane was transferred at 30 V for 16 hr to optimize the transfer of the large‐sized ATM protein (MW ~ 350kD). The membrane‐containing transferred protein was blocked with 5% nonfat milk in Tris‐buffered saline with Tween‐20 (TBST) for 1 hr. The membrane was probed overnight at 4°C with one of the primary antibodies: aggrecan (ab36861, Abcam); p53 (CST 2,524, Cell Signaling Technology); p21(sc‐397, Santa Cruz); γH₂AX (05–636, Millipore); ATM (ab2618, Abcam); p‐ATM (ab81292, Abcam); NF‐κB p65 (sc‐372, Santa Cruz); p‐p65 (CST 3,033, Cell Signaling Technology); β‐actin (ab8226, Abcam). Quantitation of protein bands was performed by densitometry analysis and local background subtraction using the ChemiDoc^TM MP system and its associated Image Lab 5.2.1 Software (Bio‐Rad, USA).

NSPCs were incubated with or without 20 μM PJ34 for 24 h. Cell lysates were prepared from 4.0 × 10⁶ cells using RIPA buffer (Nacalai Tesque). An antibody against p53 (2524, 1:500; Cell Signaling), ATM (ab2618, 1:500; Abcam), or ATR (sc-1887, 1:500; Santa Cruz Biotechnology) was added to the cell lysates, and μMACS Protein A/G MicroBeads (Miltenyi Biotec) were added to magnetically label the immune complexes. Magnetically labeled proteins were collected by using μ Columns and μMACS Separator (Miltenyi Biotec), and analyzed by immunoblotting.