Linear gradient polyacrylamide gel

Western blotting was performed as previously described.^{14 (link)} For caspase-3, PARP, and UCH-L1 detection, cell lysates were resolved on 10 or 12% SDS-PAGE. After blocking with 5% non-fat milk in TBS/Tween-20, membranes were incubated with anti-caspase-3, anti-PARP, or anti-UCH-L1 antibodies at 4 °C overnight. For Ub-protein detection, cell lysates were resolved on a 4–20% linear gradient polyacrylamide gel (Bio-Rad, Hercules, CA, USA) before incubation with anti-poly-ubiquitinated conjugates, anti-ubiquitin Lys48-specific or anti-ubiquitin Lys63-specific antibodies (1:1000 for all). Blots were washed and the appropriate secondary antibodies applied. Protein signal was visualized with ECL reagents (Pierce). Blots were subsequently stripped and re-probed using anti-GAPDH or β-actin antibodies for verification of equal protein loading. Brain cortex, hippocampus, and striatum were dissected from male 12-week-old WT and KI mice (n=4 per group) and rapidly frozen on dry ice until homogenization with T-PER tissue protein extraction reagent (Pierce) and protein measurement. Equal amounts of protein were loaded for SDS-PAGE and immunoblotted using anti-UCH-L1 antibody as described above.

Pericontusional brain cortex and hippocampus were dissected from mice at 1h, 4h or 24h after TAT-UCH-L1 treatment and injury and rapidly frozen on dry ice until homogenization with T-PER tissue protein extraction reagent (Pierce) supplemented with protease and phosphatase inhibitors. Protein concentrations were measured by BCA assay. Western blotting was performed as previously described [22 (link)]. For LC3B, Beclin-1 and HA detection, cell lysates were resolved on 10% or 12% SDS-PAGE. For Ub-protein detection, cell lysates were resolved on a 4–20% linear gradient polyacrylamide gel (BioRad, Hercules, CA) before incubation with anti-poly-ubiquitinated conjugates, anti-ubiquitin K48-specific or anti-ubiquitin K63-specific antibodies (1:1000 for all). Blots were washed and the appropriate secondary antibodies applied. Protein signal was visualized with ECL reagents (Pierce). Blots were also probed using anti-GAPDH or β-actin antibodies for verification of equal protein loading. N = 5 per group. Densitometric analysis was performed using ImageJ 1.50i software and results are normalized to the corresponding contralateral band.