Atg 5

Both tissue and cell samples were lysed using RIPA buffer supplemented with protease inhibitor cocktail (Sigma, USA). Protein concentration was measured using Bradford reagent (Sigma, USA). 20–30 μg of protein samples were loaded and separated using SDS-PAGE followed by wet transfer on a methanol charged PVDF membrane. The membrane was blocked using 5% Bovine serum albumin for an hour at room temperature and incubated with primary antibodies overnight at 4 °C. The primary antibodies used at 1:1000 concentration were mouse monoclonal hTDP-43 (Abnova, Taiwan), LC3 (Novus Biologicals, USA), Beclin-1 (Novus Biologicals, USA), p62 (Millipore, USA), ATG-5 (Millipore, USA), and Actin (Millipore, USA), GFAP (Cell signalling technologies, USA),. Subsequently, the blots were incubated with either HRP-conjugated anti-rabbit or anti-mouse secondary antibodies. Moreover, the blots were developed using ECL detection reagents and visualized with a StarBright Blue 520 (Bio-Rad Laboratories, USA). All band intensities were quantified using the ImageJ lab software. The membranes were incubated with suitable peroxidase conjugated secondary antibodies (Vector Laboratories, USA). Once the incubation was over, PBST wash was given to the blots. Blots were then treated with ECL reagent and developed using UNITECH imaging system (Cambridge) from Millipore, CA USA.

To check protein levels, the brain tissue from each group were lysed in RIPA lysis buffer containing protease and phosphatase inhibitor cocktail. To compare the level of aggregation of insoluble TDP-43 in different groups, RIPA-insoluble/soluble fractionation was performed. Further, the insoluble fraction was solubilized in 6 M urea buffer. For performing the SDS-PAGE protein concentration was evaluated using Bradford protein assay. The SDS-PAGE was performed using 20 to 25 μg total protein extract from each sample. Further, the separated protein extract was transferred to polyvinylidene fluoride (PVDF) membranes. Once the proteins were transferred, the PVDF membrane was blocked using 5% BSA in PBST (0.1% Tween 20). Corresponding primary antibodies (1:1000) were used to probe mouse monoclonal hTDP-43 (Abnova, Taiwan), LC3 (Novus Biologicals, USA), Beclin-1 (Novus Biologicals, USA), p62 (Millipore, USA), ATG-5 (Millipore, USA), and Actin (Millipore, USA). Subsequently, HRP-conjugated anti-rabbit antibody or HRP anti-mouse antibody was used for the following incubation. The blots were developed with ECL detection reagents and visualized with a StarBright Blue 520 (Bio-Rad Laboratories, USA). All band intensities were quantified using the ImageJ lab software.