Page 71 - Read Online

Klugmann et al. Rare Dis Orphan Drugs J 2023;2:8 https://dx.doi.org/10.20517/rdodj.2023.05 Page 3 of 9

pathology. The post-mortem interval, which is the time between the death of the person and the time point
at which the tissue was taken, ranged between 12 and 39 h. Brain regions analyzed included motor cortex,
hippocampus, cerebellum, and brainstem (pons). Frozen tissue blocks were used for immunoblotting and
quantitative real-time PCR (qRT-PCR), and formalin-fixed, paraffin-embedded, 4 µm thin sections were
used for immunohistochemistry. Sections from the motor cortex, hippocampus, and cerebellum were cut in
the coronal plane; sections from the brainstem were cut transversally.

Antibodies
The following primary antibodies were used for immunoblotting: mouse anti-AspRS (monoclonal; Santa
Cruz Biotechnology #sc-393275; 1:500 dilution), which is specific for amino acids 170-467 of the human
AspRS protein; rabbit anti-AspRS (polyclonal; Novus Biologicals #NBP1-85937; 1:500 dilution), which is
specific for amino acids 1-135 of the human AspRS protein; rabbit anti-GAPDH (Cell Signaling #2118S;
1:5,000 dilution); mouse anti-β-Actin clone C4 (Sigma-Aldrich #mab1501; 1:10,000 dilution). Goat anti-
mouse and goat anti-rabbit HRP-conjugated secondary antibodies were obtained from Dianova
(1:10,000 dilution).

For immunohistochemistry, the following primary antibodies were used: mouse anti-AspRS (monoclonal;
Santa Cruz Biotechnology #sc-393275; 1:50 dilution) and rabbit anti-NeuN (Cell Signaling #12943S;
1:40 dilution). Goat anti-rabbit Alexa555-conjugated and goat anti-mouse Alexa488-conjugated secondary
antibodies were obtained from Thermo Fisher (1:300 dilution). Goat anti-mouse biotin-conjugated
secondary antibodies were obtained from Jackson ImmunoResearch (#111-065-003; 1:250 dilution).

TaqMan assays
The following TaqMan assays were used for qRT-PCR: DARS1 (Applied Biosystems #Hs00154683_m1) and
β-Actin (Applied Biosystems #Hs01060665_g1).

METHODS
Sample preparation for Western blotting and qRT-PCR
Before protein and RNA lysates can be prepared for Western blotting and qRT-PCR, respectively, the frozen
human brain tissue from individual brain regions needs to be homogenized in liquid nitrogen using mortar
and pestle until a fine powder is obtained. It is important to keep the tissue in liquid nitrogen at all times
during the homogenization process. When homogenization is complete and all liquid nitrogen has
evaporated from the mortar, the tissue powder is distributed into two Eppendorf tubes. The tubes are
weighed before and after filling to determine the total tissue weight. Homogenized tissue can be kept on dry
ice for further downstream applications or stored long-term at -80 °C. One tube is used to prepare a protein
lysate for Western blot analysis; the second tube is used for RNA isolation for qRT-PCR.

Western blotting
For Western blotting, the homogenized tissue powder is resuspended in 10 µL lysis buffer [50 mM Tris-
HCL pH 7.4, 1 mM EDTA pH 8.0, 250 mM NaCl, 1% v/v Triton-X, and protease inhibitors (Roche
Complete #04693124001)] per 1 mg of tissue weight. Following sonication using a probe sonicator (Branson
Digital Sonifier 450) at 10% sonication amplitude for 1 min, the tissue suspension is spun down at full speed
in a conventional benchtop centrifuge and the supernatant is transferred to a new tube. The protein
concentration of the lysates is determined by Bradford protein assay using Quick Start Bradford 1x Dye
Reagent (Bio-Rad #500-0205). 30 µg of protein from each brain region is mixed with five times sample
buffer (15 g SDS, 15.6 mL 2M Tris pH 6.8, 57.5 g glycerol, 16.6 mL β-mercaptoethanol) and loaded onto a
10% acrylamide gel (Bio-Rad Mini-PROTEAN gel system). Following separation by SDS-PAGE, proteins
are transferred onto a PVDF membrane (Bio-Rad #162-0177). To avoid unspecific antibody binding, the