The
human neuroblastoma cell line SK-N-SH (ATCC HTB-11) was cultured
in T25 flasks using the essential modified Eagle’s medium (EMEM)
supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO), 1% Pen/Strep,
1% nonessential amino acids (Sigma-Aldrich), and 1% sodium pyruvate
(Sigma-Aldrich) and incubated at 37 °C, 5% CO2. This
neuronal cell line was chosen for this study as it has been used in
previous studies and behaves similarly to primary neuronal cells with
respect to CATB secretion and neurotoxicity.13 (link),15 (link),18 (link),21 (link) The SK-N-SH
culture medium was changed every 2–3 days until 80% confluence.
The neuronal cells were exposed to 13 dpi serum-free MCM diluted at
1:4 in EMEM and treated with the monoclonal anti-CATB antibody (8.33
μg/mL; Sigma-Aldrich), monoclonal anti-SAPC (14.1 μg/mL;
Abcam), or CA-074 CATB inhibitor (10 μM, Sigma-Aldrich) for
24 h at 37 °C, 5% CO2. The treated neuronal cells
were washed twice with sterile phosphate-buffered saline and detached
by incubation using radioimmunoprecipitation assay buffer (Abcam,
Cambridge). The cell lysates were collected and stored at −80
°C for TMT labeling experiments.
human neuroblastoma cell line SK-N-SH (ATCC HTB-11) was cultured
in T25 flasks using the essential modified Eagle’s medium (EMEM)
supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO), 1% Pen/Strep,
1% nonessential amino acids (Sigma-Aldrich), and 1% sodium pyruvate
(Sigma-Aldrich) and incubated at 37 °C, 5% CO2. This
neuronal cell line was chosen for this study as it has been used in
previous studies and behaves similarly to primary neuronal cells with
respect to CATB secretion and neurotoxicity.13 (link),15 (link),18 (link),21 (link) The SK-N-SH
culture medium was changed every 2–3 days until 80% confluence.
The neuronal cells were exposed to 13 dpi serum-free MCM diluted at
1:4 in EMEM and treated with the monoclonal anti-CATB antibody (8.33
μg/mL; Sigma-Aldrich), monoclonal anti-SAPC (14.1 μg/mL;
Abcam), or CA-074 CATB inhibitor (10 μM, Sigma-Aldrich) for
24 h at 37 °C, 5% CO2. The treated neuronal cells
were washed twice with sterile phosphate-buffered saline and detached
by incubation using radioimmunoprecipitation assay buffer (Abcam,
Cambridge). The cell lysates were collected and stored at −80
°C for TMT labeling experiments.
