Western blot analysis

For in vivo analysis, 5-mm tissue of ipsilateral cortex around the injury area from TBI mice or the same region and volume from sham mice were dissected and processed as described [⁸]. Cell lysates were prepared by lysing the H4 neuroglioma, HeLa and rat cortical neuron cultured in 24-well plates with SDS-PAGE buffer. Cell or tissue lysates were resolved on 4–20% SDS-PAGE gels (Bio-Rad Laboratories, 5671095) and transferred to PVDF membrane (Millipore Sigma, IPVH00010) using semi-dry transfer (Bio-Rad Laboratories). Membranes were blocked with 5% nonfat milk in TBST (Tris-buffered saline with 0.05% Tween 20 [National Diagnostics, 9005–64–5]), probed with primary antibodies in 1% BSA in TBST overnight at 4°C and incubated with HRP-conjugated secondary antibodies (KPL, 474–1506, 474–1806, 14–16–06 and 14–13–06) in blocking solution at room temperature for 1 h. Protein bands were detected using SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific, 34076) or SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, 34080) and visualized using Chemi-doc system (Bio-Rad Laboratories, Universal Hood II). Band intensity was analyzed using Image Lab software (Bio-Rad Laboratories) and normalized to loading control.

Primary antibodies: LC3 (1:1000; Novus Biologicals, NB100-2220), PIK3C3/Vps34 (1:1000; Invitrogen, 382100), BECN1/beclin1 (1:1000; Santa Cruz Biotechnology, sc-11427), SQSTM1 (1:1000; BD Bioscience, 610832), ATG5 (1:1000; Sigma-Aldrich, A0731), ACTB/β-actin (1:10,000; Sigma-Aldrich, A1978), phospho-PLA2G4A (1:1000; Sigma-Aldrich, SAB4503812), LAMP1 (1:1000; Abcam, 24170), PLA2G4A (1:1000; 2832), ULK1 (1:1000; Cell Signaling Technology, 4773), phospho-UK1(1:1000; Cell Signaling Technology, 5869) and α-tubulin (1:500; AA4.3-s, developed by Walsh, C. and obtained from Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52,242).