Anti rabbit hrp secondary antibodies

Urea-solubilized neuron lysates were mixed 1:1 with 2× NuPAGE LDS sample buffer and boiled for 10 min. About 30 μg of each sample were run on 12% Bis–Tris gels and transferred to polyvinylidene fluoride membranes. The blots were probed with primary antibodies including antitotal tau (1 μg/ml; tau12) and antitotal α-synuclein (1:1000 dilution; Syn211). Detergent-extracted samples were probed using polyclonal antibodies generated in-house from a rabbit immunized with a synthetic peptide corresponding to the R2 region of tau (4R specific). Anti-mouse-HRP or anti-rabbit-HRP secondary antibodies were used at 1:10,000 dilution (Thermo Fisher Scientific). Mouse anti-actin-HRP (1:2000 dilution; Novus) was used as a loading control. For detection of biotinylated proteins, the blots were blocked with 2.5% bovine serum albumin in Tris-buffered saline plus Tween-20, probed with streptavidin-HRP (1:25,000 dilution; Abcam; catalog no.: ab7403) for 1 h, blocked with 10% fetal bovine serum, and then washed with Tris-buffered saline plus Tween-20 prior to developing.

Hamster and mouse tissues were homogenized mechanically in RIPA lysis buffer supplemented with 1× protease inhibitor cocktail set (Millipore) and loaded with SDS dye. Protein concentration was measured using the Bradford assay and 60 μg of protein was used per lane. Proteins were separated on 6% polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane (Millipore) using semi-dry blotting. The membrane was blocked in 5% skim milk in TTBS, MOV10L1 was detected using anti-MOV10L1 primary antibodies^{4 (link)} (gift from J. Wang) diluted 1:250 and incubated overnight at 4°C. Anti-rabbit-HRP secondary antibodies (Thermo Fisher Scientific) were diluted 1:50,000 and the signal was detected using SuperSignal West Femto Substrate (Thermo Fisher Scientific). For TUBA4A detection, samples were run on 10% polyacrylamide gel and incubated with anti-tubulin (Sigma-Aldrich, T6074) mouse primary antibodies diluted to 1:10,000 and anti-mouse-HRP secondary antibodies (Thermo Fisher Scientific) diluted to 1:50,000.