Anti ubiquitin

Proteins were resolved on 8%, 10%, 15%, or 4-20% tris-glycine gels, and for amyloid-β or Ub immunoblotting 16% tris-tricine gel was used^{62 (link)}, transferred to nitrocellulose membranes (GE Whatman, 0.2 µm Pore Size). Western blot images were visualized by enhanced chemiluminescence (ECL). The images were captured by using an ImageQuant™ LAS 4000 imaging system (GE Healthcare) and quantitated by Image Studio™ Lite (LI-COR Biosciences). Primary antibodies were used at the following dilutions: 6E10 anti-amyloid-β (1:1000, Biolegend, Cat. no. 803015); anti-PSEN1 (1:1,000, Cell Signaling Technology, Cat. no. 5643); anti-β-tubulin (1:1,000, Abcam, Cat. no. AB24629); anti-UBB⁺¹ (1:1000, custom made by Sigma for our lab), anti-ubiquitin (1:2000, DAKO, Cat. no. Z0458), anti-UCH-L1 (1:1000, Abcam, Cat. no. ab8189), anti-GAPDH (1:10,000, Sigma, Cat. no. G9545), anti-β-actin (1:10,000, Santa Cruz, Cat. no. sc-47778).

Mice were deeply anaesthetized and trans-cardially perfused with 4% PFA. After removal of the spinal cord, spinal cord was post-fixed in 4% PFA overnight and subsequently incubated in 30% sucrose at 4 °C overnight. Thirty-five micrometers of thick free-floating sections were cut on a Leica 9000 s sliding microtome as described previously^{62 (link)} and collected in 0.1 M phosphate buffer (PB) pH 7.4. After incubation of the sections for 1 h with 4% normal goat or donkey serum and 0.3% Triton X-100 for blocking of nonspecific binding at room temperature, sections were incubated overnight at 4 °C with primary antibodies in blocking solution. After three times 10 min washing in 0.25% Triton X-100 in PB at room temperature, sections were incubated in with fluorescently labeled secondary antibodies, washed again and finally mounted with Mowiol/DABCO. The following primary antibodies were used: anti-NeuN (1:1000; Millipore, MAB377, clone A60), ChAT (1:1000; Millipore, MAB144P), anti-Ubiquitin (1:500; DAKO, Z0458), anti-Synaptophysin-1 (1:500; Synaptic Systems, 101 004). Alexa-647-, Alexa-488-, and Alexa-546-conjugated secondary antibodies were from Jackson Immuno-Research Laboratories. For visualization of F-actin Alexa Flour 532 conjugated Phalloidin (Invitrogen) was used.

Rabbit polyclonal antibodies were: anti-Protein A (Sigma, St. Louis, MI), anti-ubiquitin (Dako, Carpinteria, CA), anti-Sis1 (Yan and Craig, 1999 (link)), anti-Ydj1 (Yan and Craig, 1999 (link)), and anti-Ssa1 (Lopez-Buesa et al., 1998 (link)). Mouse monoclonal antibodies used were anti-Flag tag (M2; Sigma), anti-HA tag (12CA5; Roche, Germany), anti-Rpl3 (a gift of J. Warner), anti-GFP (Roche) and anti-Pgk1 (Invitrogen, Carlsbad, CA]). Secondary antibody was HRP-conjugated (Molecular Probes, Eugene, OR). Doxycycline hydrochloride was from Fischer Scientific (Waltham, MA). MG132 was from Cayman Chemical (Ann Arbor, MI). The recombinant catalytic core of Usp2cc was a generous gift of R. Kopito (Kaiser et al., 2011 (link)).

Co-transfected Neuro-2a cells were lysed and total protein was extracted with sonication (10 s, Setting 3, Branson Sonifier 450) in extraction buffer (1% (v/v) Nonidet P-40 in TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl) with protease inhibitor cocktail). Cellular debris was pelleted at 18,000g (30 min at 4 °C). Protein concentration was estimated using the BCA Protein Assay Reagent (Pierce Biotechnology). Typically, 3 μg of anti-ubiquitin (Dako) per 500 μg of protein extract was used for immunoprecipitations. Protein A/G magnetic beads (Pierce Biotechnology) were used to capture the antibody:protein complex. Immunoprecipitates were washed with TBS+1% (v/v) NP-40 (3 × ) to remove non-specifically bound proteins, and then resuspended in 1 × LDS buffer with 50 mM DTT, and heated at 95 °C for 10 min.