0.2 μm pvdf membrane

Whole mouse brains were dissected after isoflurane inhalation and rapid decapitation. Brains were homogenized (10 mM HEPES pH 7.4, 150 mM KCl, 5 mM MgCl₂, 0.5 mM DTT, supplemented with 1 μl rRNAsin (Promega) and 1 μl Superasin (Ambion) per ml and 1 mini EDTA-free protease inhibitor tablet (Roche) per 10 ml) using a glass homogenizer and Teflon pestle with a drill. Lysates were cleared by centrifugation at 2000 × g for 10 min. Concentration of the supernatant was determined by BCA assay (Thermo Fisher). 50 ug of total lysate was run on 4–12% SDS-PAGE gel (Bio-Rad) and transferred to 0.2 μm PVDF membrane (Bio-Rad). Ponceau image was quantified for loading control. Membranes were blocked in 5% Milk in 1X TBS with 0.5% Tween for 3 h at room temperature. Membranes were incubated in antibody overnight at 4 °C (QKI-6, AB9906/Millipore, 1:1000). Appropriate HRP conjugated secondary antibodies were applied after washes at room temperature for 1 h. Membranes were incubated in Clarity (Bio-Rad) chemiluminescence reagents for 5 min and then developed for 1 min in a myECL Imager (Thermo). N is represented in figure legend.

Cells were first lysed in HEPES buffer (pH 7.0) that contained 1% NP-40 or Glo Lysis Buffer (Promega) with protease inhibitor cocktail (Roche). Next, ∼20–60 μg of total proteins (as determined by BCA assay kit) was resolved by PAGE on a 10% Tris-glycine SDS-gel, transferred onto a 0.2-μm PVDF membrane (Bio-Rad, Hercules, CA, USA), and probed with Abs as indicated in the figures.

AβO_1–42 were prepared as described above and resolved on a nonreducing 4–15% tris-glycine–SDS-PAGE gels with LDS sample buffers [75 (link)]. The gel was transferred on to a 0.2-μm PVDF membrane (Bio-Rad) according to the manufacturer’s recommendation. Membranes were blocked in 5% bovine serum albumin (BSA) in tris-buffered saline containing 0.01% Tween 20 for 1 h at room temperature. Blots were incubated in the primary antibody mOC64 (rabbit monoclonal against amino acid residues 3–6 of Aβ; Cat# ab201060, Lot# GR3235744-4, RRID: AB_2818982, Abcam) [76 (link)] at 1:200 dilution overnight at 4 °C. Immunoreactivity was detected with enhanced chemiluminescence (Bio-Rad) and imaged using Fluorchem E system (ProteinSimple). Molecular weight values were estimated using Precision Plus Protein™ Dual Color Standards (Bio-rad).

The cells were lysed in equal volumes of ice cold lysis buffer with protease inhibitor cocktail. Cell lyses were separated by SDS-PAGE and then transferred to a 0.2-μm PVDF membrane (Bio-Rad, USA). After being blocked with Odyssey Blocking Buffer (Li-COR Biosciences, USA), the membrane was incubated with primary antibody (1:1000) at 4 °C overnight, followed by incubation with IRDye 800CW or 680 secondary antibodies (1:5000, LI-COR Biosciences, USA). ACTIN was used as endogenous control. The Odyssey Infrared Imaging System was used to visualize targeted protein bands.

A RIPA buffer (pH 7.4), containing 20 mM of Tris, 0.25 mM of NaCl, 11 mM of EDTA, 0.5 % NP-40, 50 mM of sodium fluoride, and protease, phosphatase inhibitors (Sigma, Poland) was used for homogenization of the brain samples (Yant et al. 2003 (link)). The total protein concentrations were determined, using the Bradford Protein Assay (Sigma, Poland) and the homogenates were subjected to SDS-polyacrylamide gel electrophoresis and assessed for the expression of D1 and D2 receptors. Briefly, the extracted proteins (20 μg/well) were separated on 12 % gel (SDS-PAGE), using a Mini Protean Tetra Cell System (Bio-Rad, Poland). The fractionated proteins were transferred onto a 0.2-μm PVDF membrane (Bio-Rad, Poland), next membranes were blocked with 3 % bovine serum albumin (BSA) solution in buffer for 1 h at room temperature. The brain protein expression was identified, using antibody against D1-rabbit polyclonal (SantaCruz Biotechnology, cat no sc-14001), D2-mouse monoclonal (SantaCruz Biotechnology, cat no sc-5303), GFAP-mouse monoclonal (SantaCruz Biotechnology, cat no sc-33673), and appropriate sAb bovine anti-rabbit/anti-mouse IgG HRP (Santa Cruz Biotech, USA);
The membranes were processed with an ECL Advance Western Blotting Detection Kit (Amersham Life Sciences, UK) and, subsequently, bands were visualized, using the Gel DOC-It Imaging system.

Total protein was extracted using extraction buffer (25 mM Tris/HCl pH 7.6, 15 mM MgCl₂, 150 mM NaCl, 15 mM p-nitrophenyl phosphate, 60 mM β-glycerophosphate, 0.1% NP-40, 0.1 mM Na₃VO₄, 1 mM NaF, 1 mM PMSF, 1 μM E64, complete proteinase inhibitor (Roche), 5% ethylene glycol) and the protein concentration was determined using Bradford assay (Bio-Rad). Samples were denatured in Laemmli buffer, run on a 4–15% TGX gel (Bio-Rad) for 20 min at 300 V, and subsequently blotted on a 0.2 μm PVDF membrane (Bio-Rad). Antibodies used were anti-HA (1:1,000 dilution, 3F10, Roche) and anti-actin8 (1:2,000 dilution, A0480, Sigma). Chemiluminescent detection was performed with Western Bright ECL (Isogen, http://www.isogen-lifescience.com/).

Cells were lysed on ice using RIPA buffer (Alfa Aesar) with protease inhibitor cocktail (Thermo Fisher Scientific). After protein quantification, equal quantities of cell lysate samples were mixed with Laemmli buffer (Pierce) and boiled at 95°C for 5 min. Samples were run on SDS–PAGE gel and were transferred onto a 0.2-μm-PVDF membrane (Bio-Rad). The membranes were blocked using 5% non-fat milk in TBST and were blotted with anti-ACE2 rabbit mAb (SN0754; Invitrogen), anti-PIWIL2 mouse mAb (sc-377258; Santa Cruz Biotechnology), anti-GAPDH mouse mAb (GA1R; Invitrogen), or anti–β-actin rabbit pAb (4967S; Cell Signaling Technology) overnight at 4°C in 5% non-fat milk in TBST. Secondary HRP-conjugated antibodies subsequently applied including antimouse (7076P2; Cell Signaling Technology) or anti-rabbit (7074P2; Cell Signaling Technology). The membranes were probed using the ECL system (Bio-Rad) and images were developed on the Image Studio software.