Lc3a b

Whole cell lysates were prepared with RIPA lysis buffer (1% Triton X; 1% Desoxycholate, 0,1% SDS; 150 mM NaCl; 10 mM EDTA; 20 mM Tris-HCl; pH 7.5; 100.000KIE Trasylol) freshly supplemented with 2M Urea, Pefablock, 1μg/ml Leupeptin / Aprotinin, 1μg/ml Pepstatin A and 1μM Microcystin. Protein concentration was measured using the BCA protein assay (Thermo Fisher Scientific) and adjusted to equal amounts for all samples. For immunoblotting, protein lysates were separated by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes, blocked with 5% milk / TBS Tween and incubated with the following antibodies, each diluted in 5% BSA in Tris-buffered saline containing 0,1% Tween20: PARP (C-2-10; Calbiochem), Caspase 3 (8G10), cleaved Caspase 3 (5A1E), Bcl-xL (54H6), Akt, Mcl1 (D35A5), Bcl-2 (50E3), Bax, Bim (C34C5), and LC3a/b (all Cell Signaling Technology). Bcl-xL (H-5) and HSC70 (B-6) (both Santa Cruz), beta Actin (rabbit polyclonal), Noxa (EPR9735B) and Puma (EP512Y, all Abcam). Primary antibodies were detected with peroxidase-coupled secondary antibodies (Jackson). All immunoblot analyses were performed in triplicate to ensure the reproducibility of the results.

Frozen spinal cord or brain samples were thawed on ice and sonicated in 5× v/w RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 5 mM EDTA, 0.5% sodium deoxycholate, and 0.1% SDS, pH 8.0) containing protease inhibitor cocktails (Thermo Fisher Scientific). Samples were centrifuged for 30 minutes at 4 °C, 100,000 g. The supernatant was taken as RIPA-soluble fractions. The pellet was sonicated with 5× v/w RIPA buffer as above. Then, the pellet was sonicated in 2× v/w urea buffer (7 M urea, 2 M thiourea, 4% CHAPS, and 30 mM Tris, pH 8.5) and centrifuged for 30 minutes at 22 °C, 100,000 g. This supernatant was collected as the RIPA-insoluble/urea-soluble fractions. Samples were analyzed by 10% SDS-PAGE with polyvinylidene fluoride (PVDF) membranes. Following transfer to the PVDF membranes, the immunoblot was sequentially probed with antibodies that recognize pTDP-43 (mouse, Millipore, 1:800) or LC3A/B (rabbit, Cell Signaling Technology, 1:1000). The densities of the proteins were normalized to GAPDH.

Proteins (60 μg) from all sample groups were processed as previously described [92 (link)]. Proteins were separated on SDS-PAGE and subsequently transferred to nitrocellulose sheets using a semidry blotting apparatus. Sheets were saturated for 60 min at 37 °C in blocking buffer (1xTBS, 5% milk, 0.05% Tween-20), then incubated overnight at 4 °C in blocking buffer containing primary antibodies to SOD1 (1:100, Abcam), Caspase 1 (1:100, abcam), BCL2 (1:100, Santa Cruz Biotechnology, Dallas, Texas, USA), BAX (1:100, Cell Signaling Technology), LC3A/B (1:100, Cell Signaling Technology) and β-actin (1:1000, Santa Cruz Biotechnology). After four washes in TBS containing 0.1% Tween-20, samples were incubated for 30 min at room temperature with peroxidase-conjugated secondary antibody diluted 1:1000 in 1× TBS, 5% milk, 0.05% Tween-20. Bands were visualized by the ECL method. The level of recovered protein was measured using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions.