β actin

Equal amounts of protein were added to loading buffer (BioRad, Hercules, CA, USA) containing 50 μL/mL β-mercaptoethanol and heated to 98°C for 5 minutes and then loaded onto a 4–20% gradient SDS-polyacrylamide gel (BioRad) and run for 60 minutes at 130 volts. The protein was transferred to a nitrocellulose membrane over 1 hour at 400 mA and then blocked in 5% non-fat milk for 1 hour before incubation with primary antibodies. Antibodies against LC3, p62, p-p70S6K, p70S6K, pS6, S6, phospho-AMPK, AMPK, phospho-ULK1, and ULK1 (Cell Signaling, Danvers, MA, USA), PFKFB3 (Proteintech, Chicago, IL, USA), and β-actin (Sigma) were diluted 1:1,000 and incubated overnight at 4°C, with the exception of p62 and β-actin Ab, which were incubated at room temperature for 1 hour. Membranes were washed for 30 minutes in Tris-buffered saline with Tween 20 (TBS-T) (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) before addition of secondary antibodies (anti-mouse or anti-rabbit), diluted 1:10,000 in TBS-T (Sigma). ECL Western Blotting Detection Kit (Amersham/GE Pittsburgh, PA, USA) was used to develop membranes. Quantitative densitometry was performed using Image J (NIH).

For Western blot analysis 5 × 10⁶ BM-DCs were stimulated with 1 μg/ml LPS for the described time points. The pelleted cells were lysed in a buffer containing 10 mM HEPES-KOH, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM NaF, 1 mM Na3VO4, and 1 × complete™ protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) by sonification on ice for 10 s. Total protein concentration was determined by BCA (Thermo Fisher Scientific), according to manufacturer's instructions. Whole cell extracts were used for detection of Sphk1 (Abnova, Heidelberg, Germany) and β-actin (Sigma-Aldrich). The cytosolic fraction was used after pelleting the nuclear fraction by centrifugation at 13,000 × g for 10 min at 4°C to detect caspase 3 (Cell Signaling Technology, Danvers, MA) or β-actin (Sigma Aldrich). According to the first antibodies, the second antibody anti-rabbit IgG (GE Healthcare, Little Chalfont, UK) has been used. The protein bands were detected by ECL (Thermo Fisher Scientific) following the manufacturer's protocol. Quantitative evaluation was performed by densitometry using Quantity one (Bio-Rad, Hercules, CA).

Western immunoblots were performed for the quantification of UBE2A and β-actin protein in control and AD tissues using human-specific primary antibodies directed against the control protein marker β-actin (3598–100; Sigma-Aldrich, St. Louis, MO, USA) or human UBE2A (A-18; sc-10479: H-75; sc-30078; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) or (PA5-29940; Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA).

Where required, cell lysates were prepared as previously established (21 (link)). For supernatant samples, 4 × SDS loading buffer was added and samples were analyzed following the same protocol. Cell-lysate western blot samples were probed for human TN-C (N-Terminal, mab1908, Merck Millipore, California, USA), Histidine-Tag (H1029, Sigma-Aldrich), and β-actin (A5316, Sigma-Aldrich), with TN-C expression normalized to β-actin, whilst supernatant samples were analyzed for TN-C only. Mouse BALF was concentrated by trichloroacetic acid (TCA, Sigma-Aldrich) precipitation prior to analysis and TN-C expression determined using mouse TN-C antibody (N-Terminal, T3413, Sigma-Aldrich). sEV isolates were analyzed for sEV-enriched proteins cluster of differentiation 9 (CD9, sc-13118, Santa-Cruz Biotechnology, Dallas, USA) and flotillin-1 (ab13493, Abcam, Cambridge, UK), with the negative control glucose regulated protein 94 (GRP94, ab7291, Abcam) utilized to confirm the lack of cellular protein contamination. Densitometrical analysis was performed using ImageJ software (Version 1.5i; NIH). Due to multiple variants of TN-C being expressed, the dominantly expressed band was measured for each experiment.

β-actin (Sigma Aldrich mouse monoclonal anti-β-actin, A2228, 1:2000), 42 kDa. N-cadherin (BD Biosciences mouse monoclonal anti-N-cadherin, 610921, 1:2000) 120 kDa. E-cadherin (BD Biosciences mouse monoclonal anti-E-cadherin, 610181, 1:2000) 120 kDa. Vimentin (Sigma-Aldrich goat polyclonal anti-Vimentin, V4630, 1:1000) 58 kDa. S100A10(BD Biosciences mouse monoclonal anti-S100A10, 610070, 1:2000) 11 kDa. Annexin A2 (BD Biosciences mouse monoclonal anti-Annexin II, 610069, 1:2000) 36 kDa. GAPDH (Biochain mouse monoclonal anti-GAPDH, Y3322, 1:2000) 36 kDa. p-S6K (Cell signaling rabbit monoclonal anti-pS6K, 9205 S, 1:1000) 70 kDa. FOXC2 (Bethyl laboratories rabbit polyclonal anti-FOXC2, A302-383A, 1:1000) 65–70 kDa. PAI-1 (Cell signaling rabbit monoclonal anti-PAI-1 D9C4, 11907, 1:2000) 48 kDa. uPAR (Santa Cruz rabbit polyclonal anti-uPA H149, sc-10815, 1:300) 55 kDa. p-ERK (Erk1/2) (Cell signaling rabbit polyclonal anti-Erk1/2 (Thr202/Tyr204), 9101, 1:1000) 42, 44 kDa.

Cells dislodged from flasks in PBS with 0.6 mM PMSF were centrifuged at 1,500 g for 10 min, suspended in 50 mM Tris–HCl pH 8.9 and sheared by bath sonication. All antibodies were from Santa Cruz except β-actin (Sigma) at the following concentrations: PrP (clone 6D11) @1:15,000, SV40 Tag (Pab 419) @1:500, α-tubulin (clone A-6) @1:750, β-actin (clone AC-15) @1:6,000 and Annexin II (clone C-10) @1:500. All these primary mouse antibodies were detected using m-IgG-kappa BP HRP @1:800 as the secondary antibody. For quantitation, chemiluminescent signals on blots were normalized with respect to protein loaded/lane and additionally confirmed by β-actin, α-tubulin, and Annexin II.