I block

Protein was isolated from samples using Trizol (Invitrogen). Protein concentration was measured using the BCA kit (Thermo Scientific) and 40 μg of protein/lane loaded in a 10% SDS PAGE precast gel (Invitrogen). Gel was transferred using the iBlot transfer system (Invitrogen). Nitrocellulose membrane was blocked in 2% I-Block (Applied Biosystems) in 1×TBS-T for one hour, and then either b-actin (Abcam, 1:10,000), Histone H3 (Cell Signaling, 1:1000), RAGE (R&D Biosystems, 1:500), TLR4 (Santa Cruz, 1:500), or NFκB (Abcam, 1:1,000) was used. Secondary antibodies (anti-mouse, Jackson ImmunoResearch) were added at 1:1,000 dilution in 2% I-Block in 1×TBS-T on a room temperature. The membranes were washed with 1×TBS-T, and then Luminol Reagent (Santa Cruz) was added. The membranes were then developed using a FluorChem E Imager system (ProteinSimple).

Protein was extracted from samples using Trizol (Invitrogen). The BCA kit (Thermo Scientific) was used to measure protein concentration and 40 μg of protein/lane loaded in a 10% SDS PAGE precast gel (Invitrogen). The gel was placed in a tank where 120 volts of electrical current was run for approximately one and a half hours. The gel was subsequently transferred to a nitrocellulose membrane using the iBlot transfer system (Invitrogen). This membrane was blocked in 2% I-Block (Applied Biosystems) in 1× TBS-T for 1 h, and then either β-actin (Abcam, 1:10,000), TGF-β (R&D Sytems, 1:1,000), Amyloid-β 1–40 (MyBioresource, 1:500) or Amyloid-β 1–42 (Abcam, 1:1,000) was used. For Synaptophysin and PSD-95, 30 μg of protein/lane was loaded, and either GAPDH (Abcam, 1:5,000), PSD-95 (Cell signaling, 1:750) or Synaptophysin (Chemicon, 1:2,000) were used. Secondary antibodies (anti-mouse, Jackson ImmunoResearch) were added at 1:1,000 dilution in 2% I-Block in 1× TBS-T at room temperature. The membranes were washed with 1× TBS-T, and then Luminol Reagent (Santa Cruz) was added. The membranes were then developed using a FluorChem E Imager system (ProteinSimple). Grayscale images were analyzed using ImageJ and normalized to β-actin or GAPDH.