Neuronal protein extraction reagent

Brains were harvested, weighed, and one hemisphere of each brain (not including the midbrain, pons, or medulla) was isolated for TNFα western blot analysis. Brains were suspended in 1 ml Neuronal Protein Extraction Reagent (Thermo Scientific, 87792) buffer with added protease inhibitors (Roche complete mini protease inhibitor cocktail tablets, Sigma, 4693124001, 1:10). Tissue was homogenized and incubated for 10 min on ice. Samples were centrifuged at 10,000g for 10 min and lysates were removed. The Pierce BCA Protein Assay Kit was used to measure the supernatant protein concentration. Tissue samples were separated by SDS‐PAGE and transferred to a nitrocellulose membrane. The membrane was blocked in 5% nonfat milk for 1 h, incubated overnight in primary TNFα (1:1000, Cell Signaling 11948, RRID:AB_2687962) antibody, and washed with TBS‐Tween*‐20 (Table S1). Subsequently, the membrane was incubated in secondary antibody (1:4000, anti‐rabbit IgG, Bio‐Rad 170‐6515, RRID:AB_11125142) in 5% nonfat milk in TBS‐Tween‐20. GE Healthcare ECL prime western blotting detection reagent was used and blots were imaged with a CCD camera. TNFα and GAPDH (1:20,000, Sigma, RRID:AB_796208) protein levels and relative densities were measured using NIH Image J software (Table S1). Background was subtracted and TNFα relative density was normalized to GAPDH.

Frozen hippocampi was placed in ice-cold buffered sucrose solution (250 μL per hippocampus), containing 10 mM Tris, pH 7.4, 320 mM sucrose, phosphatase, and proteinase inhibitors (Thermo Scientific). Samples were homogenized with a pestle and drill homogenizer. Homogenates were centrifuged for 10 min at 1,000 ×g. Supernatant (S1) was collected and transferred to fresh tube and centrifuged for 15 min at 10,000 × g. The pellet (P2), which contains synaptosomal plasma membrane, was suspended in 60 μL of Neuronal Protein Extraction Reagent (Thermo Scientific), boiled. The supernatant (S2) was centrifuged for 60 min at 100,000 × g, and the supernatant (S3) that is the cytosolic fraction was precipitated with cold acetone for 12 hr at −20°C and centrifuged for 10 min at 15,000 × g, supernatant was removed. Pellet was resuspended with 60 μL Neuronal Extraction Reagent, boiled. Protein concentrations were quantified with Pierce BCA Protein Assay Kit (Thermo Scientific) and then kept at −80°C until used.

Cell pellets of enriched cell populations from pooled pair of mouse eyes were dissolved in reducing Laemmli sample buffer, denatured and sonicated. Neuronal protein extraction reagent (Thermo Fisher Scientific, Braunschweig, Germany) was added to the neuron populations. Samples were separated on a 12% SDS-PAGE. The immunoblot was performed as previously described (Schäfer et al., 2017 (link)). Detection was performed with primary and secondary antibodies diluted in blocking solution (Table S7). Blots were developed with WesternSure PREMIUM Chemiluminescent Substrate (LI-COR, Bad Homburg, Germany). To validate specificity of the antibodies, all of them were tested on mouse serum as positive control (Figure S3).