Gapdh no 2118

Antibodies against APG7 (No. 8558), LC3A/B (No. 12741), caspase-3 (No. 9662) and GAPDH (No. 2118) were purchased from Cell Signaling Technology (Boston, MA), and horseradish peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG were purchased from Abcam (Cambridge, MA). Proteins were detected using Western blot analysis as described previously^{24 (link)}. Cellular proteins were extracted according to the manufacturer’s instructions (Santa Cruz, CA). Equal amount of proteins were separated on a 10% SDS-PAGE gel and transferred onto a PVDF membrane (Millipore,MA), which was then incubated with primary antibodies and then with secondary antibodies. The membrane was developed, and protein signals were detected using chemiluminescence. Image acquisition tools and image processing software packages used was Quantity One (provided by Bio-Rad Technology).

WB analysis was performed as described in detail previously.^{28 (link), 61 (link)} For tissue analysis, rats were killed at 24 h after SAH induction, and the cortical samples were homogenized in RIPA buffer (Sangon, Shanghai, China) and centrifuged at 12 000 × g for 15 min at 4 °C. The supernatants were collected and protein concentrations were determined with a BCA Kit (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts (60 μg) of protein per sample were subjected to WB. The antibodies used include: GCN5 (CST, no. 3305), H2B (CST, no. 2722), H3 (CST, no. 9715), H4 (CST, no. 2592), Ac-H2BK5 (CST, no. 2574), Ac-H3K9 (CST, no. 9671), Ac-H3K14 (CST, no. 7627), Ac-H3K27 (CST, no. 4353), Ac-H4K12 (CST, no. 2591), Ac-H4K5 (CST, no. 8647), Caspase 3 (CST, no. 9662), Caspase 6 (CST, no. 9762), Bim (CST, no. 3305), E2F1 (CST, no. 3742), Egr-1 (CST, no. 4154), GAPDH (no. 2118) (Cell Signaling Technology; diluted at 1:1000), Flag, or tubulin (Sigma; both diluted at 1:10 000). The horseradish peroxidase-conjugated secondary antibodies are used (Jackson ImmunoRes, West Grove, PA, USA), and signals are visualized by using the ECL chemiluminescence system (Thermo Fisher Scientific, Rockford, IL, USA).