Bis tris polyacrylamide gel

In order to analyze the expression level of EGFP-UreB and the enrichment of the TAP fusion proteins, the rabbit anti-GFP polyclonal antibody (1:1,000, GenScript) and the anti-calmodulin binding protein epitope tag antibody (1:5,000, UPSTATE) were used. The total proteins of A. fumigatus or the purified TAP fusion proteins were mixed with NuPAGE LDS sample buffer, separated on a 4 to 12% (wt/vol) Bis-Tris polyacrylamide gel (Invitrogen), and wet transferred to a PVDF membrane (Millipore). The membranes were blocked with 5% skim milk in TBST (0.05% Tween 20 in 25 mM Tris-HCl and 500 mM NaCl [pH 7.5]) for 2 h at room temperature and were then incubated for 1 h with the specific primary antibodies. The membranes were washed three times for 15 min with PBST and were further incubated with a 1:10,000 dilution of horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (EarthOx) for 1 h. After 3 washes with TBST, the protein bands were detected using ECL chemiluminescence reagents (Coolaber).

Cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS) (1x) and lysed in radio immunoprecipitation assay (RIPA) buffer (Sigma) supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific). Secreted INHBA proteins were precipitated from conditioned medium Opti-MEM using 10% (w/v) trichloroacetic acid. Protein concentration of the lysates was estimated using BCA protein assay kit (Pierce). The proteins were separated on 4–12% gradient Bis–Tris polyacrylamide gel (Invitrogen) and transferred onto a nitrocellulose membrane (GE Healthcare). The membrane was blocked by Odyssey® Blocking Buffer (Li-Cor), then incubated overnight at 4 °C with primary antibody (anti-β-actin antibody, Abcam, 1:1000; anti-INHBA, OriGene, 1:1000; anti-AKT, Cell Signaling Technology, 1:1000; Anti-phospho-AKT, Cell signaling Technology, 1:1000; anti-SMAD2, Cell Signaling Technology, 1:1000; or anti-phospho-SMAD2, Cell Signaling Technology, 1:1000). The secondary antibody used were Alexa Fluor 680 donkey anti-rabbit-IgG, IRDye 680RD donkey anti-mouse, and IRDye 800CW goat anti-rabbit. The immunoblots were imaged on the LI-COR Odyssey® 9120 Infrared Imaging system.

Mitochondrial proteins (200 µg) were solubilized in 100 µl of ice-cold 1× native buffer (Life Technologies, Inc.) containing 1% digitonin. The solubilized mitochondrial proteins were clarified by centrifugation at 100,000 × g for 30 min at 4°C. The supernatants were mixed with G250 sample additive (Invitrogen) and were electrophoresed on a precast (4% to 16%) bis-Tris polyacrylamide gel (Invitrogen), according to the manufacturer’s protocol. Protein complexes were detected by immunoblot analysis. Molecular size marker proteins apoferritin dimer (886 kDa) and apoferritin monomer (443 kDa), β-amylase (200 kDa), alcohol dehydrogenase (150 kDa), and bovine serum albumin (66 kDa) were electrophoresed on the same gel and visualized by Coomassie staining. Gel strips representing a single lane on the first-dimension native gel were excised. Gel strips were first incubated in buffer containing 1% SDS and 5% β-mercaptoethanol for 30 min to denature proteins and then electrophoresed on a second dimension using Tricine–SDS-PAGE (12%). After separation, proteins were transferred to a nitrocellulose membrane for Western blot analysis.

Samples were separated on 4–12% Bis–Tris polyacrylamide gel (Invitrogen) followed by transfer to Amersham™ Hybond® P 0.2 PVDF (GE Healthcare) for immunoblotting. Primary antibodies: anti- anti-ELP1 (1:250, Abcam, ab56362), anti-ELP3 (1:1000, Abcam, ab96781), anti-AAG (1:1000, custom rabbit polyclonal antibody, Covance, raised against ∆80AAG) or anti-AAG (1:1000, LSBio LS-C133325), anti-RNA pol II S2P (1:1000, Abcam, ab5095), anti-HA (1:1000, Abcam, ab9110), anti-α-Tubulin (1:10000, Cell Signaling, 2144), anti-H3 (1:1000, Abcam, ab1791), anti-GFP (1:1000, Abcam, ab290); were detected using infrared (IR) Dye-conjugated secondary antibodies (1:15000, Li-COR Bioscienecs, 827-11081 and 925-32210). The signal was visualized by using direct IR fluorescence via the Odyssey Scanner, LI-COR Biosciences.

Western Blot analyses of recombinant full-length 4R/0 N tau, tauΔ2–18 and soluble fractions from brain homogenates were performed to confirm the specificity and binding ability of 1C9 and Armanezumab to pathological human tau. Commercial HT7 and TNT-1 antibodies have been used as positive controls. Briefly, brain homogenates containing equal amounts of total protein in SDS sample buffer (non-reducing conditions without heat incubation) were subjected to electrophoresis in 10% Bis-Tris polyacrylamide gel in MES buffer (Invitrogen, CA), then electro-transferred onto nitrocellulose membrane (GE Healthcare, NJ). The membranes were blocked overnight with 5% fat-free dry milk in TBS with 0.05% Tween following by detection of tau using 1C9, Armanezumab, HT7 (Life Technology, CA) or TNT-1 (Millipore, MA) monoclonal antibody and appropriate HRP-conjugated secondary antibody. All primary antibodies were used at concentration 1 μg/ml. Proteins were visualized with enhanced chemiluminescence detection using Luminol reagent (Santa Cruz Biotechnology, CA).