Cleaved casp7

Various antibodies from different vendors were used in this study.
Cell Signaling (Danvers, MA): PARP (9542); cleaved PARP [Asp214, 89 kDa fragment] (9545/94885); cleaved Casp3 [Asp175, 17 kDa fragment] (9661/9664); cleaved Casp7 [20 kDa fragment] (9492); phospho-H₂AX (S139) (9718); phospho-p53 (S15) (12571); total p53 for ChIP (32532); PUMA (14570); AIF (4642); cytochrome c oxygenase IV (COX IV) (4884); Lamin A/C (4777); phospho-c-Jun (S63) (2361); phospho-c-Jun (S73) (3270); total c-Jun (9165); total c-Fos (2250). Millipore (Ontario, Canada): phospho-ATM(S1981) (05-740); Sp1 for ChIP (07-645); p21—mouse samples (188224); Milli-Mark^TM Pan-Neuronal Marker (MAB2300). BD Biosciences (San Jose, CA): p21—rat samples (556430). Sigma-Aldrich (St. Louis, MO): β-actin: A1978. Santa Cruz (Dallas, TX): cytochrome c (sc-13560). Enzo (Farmingdale, NY): α-Fodrin or αII-spectrin (a subunit of Fodrin, also known as “brain spectrin”)^{41 (link)}: BML-FG6090. Abcam (Cambridge, MA): phospho-p53 (ab1431).

Cells were lysed in cell lysis buffer (Beyotime), and the total cellular protein concentration was determined with a BCA Protein Assay Kit (Thermo Fisher Scientific Inc., IL, USA). Equal amounts of protein was separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). These membranes were probed overnight at 4°C with the following primary antibodies: antibodies against human PARP, p-ATM, p-ATR, γ-H₂AX, p-Chk1/2, Cyto c, p-JNK, ATF-2, c-Jun, MKK-4, SDHA, Casp 3, and cleaved Casp 7 (all 1:1000; Cell Signaling Technology, MA, USA), antibody against ACTB (1:2000; Zsbio, China), followed by secondary antibody (Zsbio) with peroxidase for 1 hour at room temperature.

Protein extraction from macrophages was performed using TRIzol reagent according to the manufacturer's instructions. Briefly, after phase separation using chloroform, 100% ethanol was added to the interphase/phenol–chloroform layer to precipitate genomic DNA. Subsequently, the phenol–ethanol supernatant was mixed with isopropanol to isolate proteins. The Bradford method was used to determine protein concentrations in cell lysates. Equal amounts of protein or supernatants were separated by 13.5% SDS–PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. Membranes were incubated overnight with antibodies against CASP11 (Cell Signaling, 14340), murine CASP1 (Adipogen, AG‐20B‐0042‐C100), human cleaved CASP1 (Cell Signaling Technology, 4199), human CASP1 (Cell Signaling Technology, 2225), cleaved CASP7 (Cell Signaling Technology, 9491), human CASP4 (MBL, M0293), murine IL‐1β (R&D Systems, AF‐401‐NA), and GAPDH (Cell Signaling Technology, 2118). Corresponding secondary antibodies conjugated with horseradish peroxidase and in combination with enhanced chemiluminescence reagent were used to visualize protein bands. Densitometry analyses were performed by normalizing target protein bands to their respective loading control (GAPDH) using ImageJ software as previously described 8, 9.