Anti cisd2

Total protein was extracted from cultured neuron and glial cells in a lysis buffer containing 20 mM Tris-HCl, 0.1% SDS, 0.8% NaCl, and 1% Triton-X 100. Electrophoresis with a 12% gradient was performed to separate the protein extracts. Proteins that underwent electrophoresis were electro-transferred to a nitrocellulose membrane, and then incubated with blocking reagent, primary antibodies (anti-CISD2 (1:500) (Thermo Scientific, Waltham, MA, USA, PA5-34545), and anti-GAPDH (1:500) (Millipore, Billerica, MA, USA)) for 12 h at 4 °C, before being washed and incubated with anti-rabbit IgG (HRP-conjugated secondary antibody) (Merck Millipore, Cat.#12-348) for 1 h. Results were detected using chemiluminescence (Merck Millipore, WBKLS0500). Bands of interest were visualized and quantified using the ImageQuant^TM LAS 4000 (GE Healthcare Life Sciences, Marlborough, MA, USA).

Total protein was extracted from cultured EOC microglial cells in a lysis buffer containing 20 mM Tris-HCl, 0.1% sodium dodecyl sulfate (SDS), 0.8% NaCl, and 1% Triton-X 100). Electrophoresis with 12% gradient gel was performed to separate the protein extracts. Proteins that underwent electrophoresis were electro-transferred to a nitrocellulose membrane prepared with blocking reagent and primary antibodies [anti-CISD2 (1:500; Thermo Scientific, PA5-34545); anti-iNOS (1:2,000; Thermo Scientific, PA3-030A); anti-Arg-1 (1:5,000; Thermo Scientific, PA5-29645); anti-GAPDH (1:500; Millipore, Billerica, MA, USA)] at 4°C for 12 h before washing and incubation with goat-anti-rabbit IgG HRP (horseradish peroxidase)-conjugated secondary antibodies (Merck Millipore, Cat. #12-348) for 1 h. Chemiluminescence detection was performed following the established protocols (Merck Millipore, WBKLS0500). Bands of interest were visualized and quantified using ImageQuant^TM LAS 4000 (GE Healthcare Life Sciences, Marlborough, MA, USA).

Total protein was extracted from cultured EOC microglial cells or animal tissues in a lysis buffer containing 20 mM Tris-HCl, 0.1% sodium dodecyl sulfate, 0.8% NaCl, and 1% Triton-X 100. The proteins were resolved using a 12% gradient gel. The resolved proteins were electro-transferred to a nitrocellulose membrane. The membrane was blocked with a blocking reagent and incubated with the following primary antibodies at 4 °C for 12 h: anti-CISD2 (1:500; catalog: PA5-34545; Thermo Scientific, Waltham, MA, USA); anti-iNOS (1:2000; catalog: PA3-030A; Thermo Scientific); anti-Arg1 (1:5000; catalog: PA5-29645; Thermo Scientific); anti-GAPDH (1:500; catalog: #MAB374; Millipore, Billerica, MA, USA); and anti-α tubulin 4a (1:1000; catalog: GTX112141; Hsinchu, Taiwan); anti-β-actin (1:4000; Catalog: ab8227; Abcam). Next, the membrane was washed and incubated with goat anti-rabbit IgG HRP-conjugated secondary antibodies (catalog: #12-348, Millipore) for 1 h. Immunoreactive signals were detected using a chemiluminescence detection kit (Catalog: WBKLS0500, Millipore). The target bands were visualized and quantified using ImageQuant^TM LAS 4000 (GE Healthcare Life Science, Marlborough, MA, USA).