Pierce ecl plus

Western blot analyses was performed on brain tissue collected from one non-inoculated, one TME-infected, and eight BSE-infected cattle: two classified as C-type, four as H-type, and two as L -type. Each sample was separated by SDS-PAGE on 12% polyacrylamide minigels (Invitrogen) and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA) for 45 min at 30 V. The membranes were blocked with 3% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for 1 h and incubated at 4°C overnight with mouse anti-PrP monoclonal antibody 6H4 at a 1:10,000 dilution (0.1 μg/ml) as the primary antibody. Then, a biotinylated sheep anti-mouse secondary antibody at 0.05 μg/ml and a streptavidin–horseradish peroxidase (HRP) conjugate, were used in conjunction with a chemiluminescent detection system (Pierce ECL plus, Thermo Fisher) and visualized on an imaging system capable of detecting luminescence.

Cell lysates (20 μg total protein) as described^{34 (link)} were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to PVDF membrane. After blocking, membranes were incubated overnight at 4  °C with primary antibodies against Cx26 (Invitrogen, Grand Island, NY, USA), Cx43 (Cell Signaling, Danvers, MA, USA), NANOG (Cell Signaling, Danvers, MA, USA), GFP (Invitrogen, Grand Island, NY, USA), SOX2 (Cell Signaling, Danvers, MA, USA), OCT4 (Cell Signaling, Danvers, MA, USA), phospho-FAK (Y397, Y576, Y925) (Cell Signaling, Danvers, MA, USA), total FAK (Cell Signaling, Danvers, MA, USA), and/or β-actin (Santa Cruz, Dallas, TX, USA), followed by incubation with secondary anti-mouse or anti-rabbit immunoglobulin G (IgG) antibodies conjugated to horseradish peroxidase (Thermo Scientific, Waltham, MA, USA). Immunoreactive bands were visualized by exposing films to luminescent signals generated after incubating the membrane with Pierce ECL plus (Thermo Scientific, Waltham, MA, USA). Copies of the uncropped blot scans are provided in the supplementary information (Supplementary Figs. 14–38) with associated molecular weights indicated for each antibody.

Protein was extracted from cells using RIPA buffer (89900, Thermo Fisher Scientific). A total of 60 µg of protein (from whole cell extract) was separated by electrophoresis in a 4–15% precast protein gel (4561086, BioRad) and transferred to PVDF membranes. Blocking was subsequently performed with 5% non-fat milk in TBST, followed by incubation with anti-H3K27Ac antibody at 1:500 dilution (8173S, Cell Signaling Technology) overnight. After 5 washes with TBST, membranes were incubated with HRP-conjugated anti-Rabbit IgG antibody at 1:1000 (7074 Cell Signaling Technology) for 1 h. Pierce ECL Plus (32132, Thermo Fisher Scientific) was used to detect protein bands. Blots were then stripped (46430, Thermo Fisher Scientific) and re-probed with anti-total H3 primary antibody at 1:1000 dilution (14269S, Cell Signaling Technology) as a loading control. HRP-conjugated anti-Mouse IgG antibody (7076, Cell Signaling Technology) was used to detect total H3 signal. Densitometry analysis was performed with image J.

Brain slices were stimulated with DHPG and were collected (2 slices per sample) in radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS). The samples were clarified by centrifugation at 14,000× g and the protein concentration was determined using a bicinchoninic acid assay kit (BCA; Thermo Scientific, Merelbeke, Belgium). The samples were heated for 10 min at 70 °C. A total of 10 μg of protein for each sample was loaded on a 7% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking with 5% non-fat milk, the membranes were probed overnight with anti-Arc (1/1000, Cell Signaling, Leiden, The Netherlands), anti-phospho-p44/42 MPK (pERK1/2, 1/1000, Cell Signaling Technology), anti-spectrin (1/1500, Merck Millipore, Overijse, Belgium), anti-B56α (1/250, Santa Cruz Biotechnology, Dallas, TX, USA), anti-GluA1 (1/500, Merck Millipore), anti-GluA2 (1/1000, Merck Millipore), anti-GAPDH (1/1000, Cell Signaling Technology), or anti-β tubulin (1/1000, Neuromics, Edina, MN, USA). The membranes were then incubated with secondary antibodies coupled to peroxidase (Dako, Agilent Technologies, Diegem, Belgium) and peroxidase was detected with Pierce ECL Plus (Thermo Scientific) on ECL hyperfilm.

Proteins were resolved by SDS-PAGE on NuPage 4%–12% Bis-Tris gels (Invitrogen) and transferred to a nitrocellulose membrane (Bio-Rad). The blots were blocked in blocking buffer (5% milk in TBS-T) and then incubated with primary antibodies in blocking buffer overnight at 4°C. After 3 washes (5 minutes each) with TBS-T, the blots were incubated with HRP-conjugated secondary antibodies in blocking buffer for 1 hour at RT, washed again for 3 times (5 minutes each) in TBS-T and developed using ECL-based detection (Pierce ECL plus, Thermo Scientific). The following primary antibodies were used for western blot analysis: mouse anti-Rpl19 (Abcam, Cat#ab58328; RRID: AB_945305, 1:1000), mouse anti-Rps23 (Abcam, Cat#ab57644; RRID: AB_945314, 1:1000), rabbit anti-Rps4X (Proteintech, Cat#14799-1-AP; RRID: AB_2238567, 1:1000), rabbit β-catenin (Sigma-Aldrich, Cat#C2206; RRID: AB_476831, 1:8000) and rabbit anti-β-actin (Abcam, Cat#ab8227; RRID: AB_2305186).

Total protein from cells or tissues was harvested with ice-cold RIPA buffer (10 mM Tris, 150 mM sodium chloride, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1% NP-40, pH 7.4) plus protease inhibitor cocktail (Thermo Fisher Scientific) and phosphatase inhibitor cocktail (Roche). Equivalent amounts of protein were separated by 10% or 12% SDS–PAGE and transferred onto a 0.22-mm PVDF membrane (Merck Millipore, Burlington, MA, USA). The membranes were blocked with 5% skimmed milk (w/v) in TBST buffer for 1 h and incubated with the rabbit polyclonal to AGE antibody (Abcam, 1:1000) with gentle rocking overnight at 4°C. The membranes were further incubated with goat anti-rabbit HRP-conjugated secondary antibodies (Cell Signaling Technology, 1:1500) for 1.5 h at room temperature. Finally, protein bands were imaged using an enhanced chemiluminescence detection kit (Pierce ECL Plus, Thermo Scientific) and analyzed with a luminescent image analyzer (ImageQuant LAS 4000 or Amersham Imager 600, GE Healthcare Life Sciences).