Ecl solution

Ctrl (n = 5), C9orf72 (n = 3), FUS (n = 1), TARDBP (n = 2), and uvALS (n = 3) fibroblasts were lysed on plates in 2xLaemmli buffer and the lysates were boiled at 100 °C for 5 min. Spinal cords, sciatic nerves, and gastrocnemius muscles of n = 4 animals per group were dissected [1 (link)] and lysed in homogenization buffer (50 mM Tris HCl pH 7.4, 250 mM NaCl, 1 mM EDTA, 5 mM MgCl₂, 1% Triton X-100, 0.25% Na-deoxycholate, 0.1% SDS, protease inhibitor cocktail from Sigma-Aldrich). After 2 × 10″ sonication cycles, samples were incubated on ice and then centrifuged at 15,000×g for 20′ at 4 °C. Supernatants were then quantified with Bradford protein assay (Bio-Rad) and resuspended in Laemmli Buffer before SDS-PAGE (Sigma-Aldrich). Proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes, followed by incubation with 5% skimmed milk for 1 h and with primary antibodies at 4 °C overnight. HRP-conjugated secondary antibodies (1:2,500, Jackson ImmunoResearch) were applied at RT for 1 h. ECL solution (Roche) was used for chemiluminescent detection. GAPDH was used as a control for equal loading. Following densitometry-based quantification and analysis using ImageJ software, the relative density of each identified protein was calculated.

Western blot analysis was done to evaluate apoptosis status by monitoring protein levels of Bax and Bcl-2 in HUVECs from different groups. For protein lysis, cells were collected and lysed in ice-cold cell lysis buffer solution contained protease inhibitor cocktails (Sigma) and centrifuged at 14000 rpm for 20 minutes at 4°C. Total protein content was determined in the supernatant by using the PicoDrop spectrophotometer (Model: PICOPET01). To resolve protein samples, 100 μg of cell lysate was loaded in the lanes of 12% SDS-polyacrylamide gel and then transferred to PVDF membrane (Millipore). The membranes were incubated with specific antibodies against Bcl-2 and Bax (Dilution: 1:1000, both from Abcam) at 4°C overnight. The next day, blots were incubated with the HRP-conjugated anti-rabbit IgG antibody for 1 h (Abcam). The immune-reactive bands were determined by using the ECL solution (Roche). Using the HP ScanJet G31110 system, we scanned the x-ray films. Densitometry evaluation of bands was done with ImageJ software ver.1.44p. We used β-actin as an internal loading control.