APOE4-R47H Neuron Manuscript (Carling et al.)
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Unprocessed western blot source data for Carling et al., 2024 Neuron manuscript.
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For mouse brain samples, hippocampal or frontal cortex tissue was mechanically homogenized on ice in RIPA buffer (Thermo Fisher Scientific, 89901) supplemented with Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific, 78420) and cOmplete protease inhibitor cocktail (Roche, 11697498001). After sonication, brain lysates were centrifuged at 20,000 g for 15 minutes at 4C. Supernatants were collected, and protein concentration was measured by BCA (Pierce, 23225). For cultured primary microglia, cells were plated at a density of 500,000 cells/well in a PDL-coated 6-well plate in growth media. The next day, cells were treated with 1 g/ml of 0N4R tau fibrils (from Dr. Sue-Ann Mok, University of Alberta) per well for 24 hours. Cells were lysed in RIPA buffer supplemented with Halt phosphatase inhibitor cocktail and cOmplete protease inhibitor cocktail. Samples were rotated for 15 minutes at 4C, and lysates were cleared by centrifugation at 20,000 g for 10 minutes at 4C. Protein concentration was measured by BCA. 50 g of brain lysates or 10 g of primary microglial lysates were evenly loaded based on protein concentration onto NuPAGE Bis-Tris gels (Invitrogen) and run in MES SDS running buffer (Invitrogen, NP0002) at 130 volts for approximately 1 hour. Gels were transferred to nitrocellulose membranes (Bio-Rad, 1620115) at 0.25 amps for 2 hours at 4C. Membranes were washed three times for 10 minutes each in TBS with 0.01% Triton X-100 (TBST) and were blocked for 1 hour in 5% milk in TBST. Primary antibodies were diluted in 1% milk in TBST and incubated at 4C overnight. The following day, membranes were washed three times for 10 minutes each with TBST and incubated with appropriate secondary antibodies in 1% milk in TBST for 1 hour at room temperature. Membranes were washed again three times for 10 minutes each in TBST to minimize non-specific binding. Membranes were then treated with ECL (Bio-Rad, 1705060) for 60 seconds and developed using a Bio-Rad imager. Blots were scanned at 300 d.p.i. and quantified using Image Lab (Bio-Rad). Primary antibodies used for western blot were anti-HT7 (Thermo Fisher Scientific Cat# MN1000, RRID:AB_2314654, 1:1000 dilution), anti-AT8 (Thermo Fisher Scientific Cat# MN1020, RRID:AB_223647, 1:1000 dilution), anti-cGAS (Cell Signaling Technology Cat# 31659, RRID:AB_2799008, 1:500 dilution), anti-STING (Cell Signaling Technology Cat# 50494, RRID:AB_2799375, 1:500 dilution), anti-TREM2 (Cell Signaling Technology Cat# 91068, RRID:AB_2721119, 1:500 dilution), anti-APOE (Millipore Cat# 178479, RRID:AB_10682965, 1:500 dilution), and anti--Actin (Sigma-Aldrich Cat# A3854, RRID:AB_262011, 1:10000 dilution). Immunoreactivity was detected with goat anti-rabbit HRP (Jackson ImmunoResearch Labs Cat# 111-035-144, RRID:AB_2307391, 1:5000 dilution), goat anti-mouse HRP (Jackson ImmunoResearch Labs Cat# 115-035-146, RRID:AB_2307392), or rabbit anti-goat HRP (Thermo Fisher Scientific Cat# 81-1620, RRID:AB_2534006).