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9 protocols using cs analyzer4

1

Immunofluorescence Quantification of Protein Colocalization

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Immunofluorescence staining was performed as described previously43 (link). Staining was visualized under a confocal microscope (Olympus FV1000D). The quantification of co-localization between OASIS and GM130 signals was calculated on a pixel-by-pixel basis WCIF version of ImageJ (http://www.uhnresearch.ca/facilities/wcif/imagej/), which generates a scatter-plot of the pixel intensities to calculate the threshold for each channel45 (link). The scatter-plot is then used to calculate the number of co-localized pixels and their intensities. To calculate the proportion of co-localization, we used the sum of intensities greater than the threshold that showed co-localization divided by the sum of intensities greater than the threshold of the respective channel that did not co-localize. Fluorescence intensity in nuclear area was measured by Image_analysis_software CS Analyzer 4 (ATTO CORPORATION).
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2

Analysis of Xbp1 Splicing by PCR

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The splicing of Xbp1 was analyzed by performing PCR in a 20 μl reaction mix containing 0.5 μm of each primer, 0.2 mM dNTPs, 3 units of Taq polymerase, and 10× PCR buffer (Agilent). The PCR condition was as follows: 94 °C for 5 min; 25 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 1 min; and 72 °C for 7 min. The PCR products were resolved by electrophoresis on a 4.8% acrylamide gel. The density of each band was quantified using Image_analysis_software CS Analyzer 4 (ATTO CORPORATION). Sequences of primers used in this study are listed in Supplementary Table.
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3

Hippocampal Protein Expression Analysis

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Homogenized hippocampal lysates were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to Immun-Blot PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were incubated with 3% bovine serum albumin (BSA) in TBST for 1 h, followed by incubation with primary antibodies against BDNF (ab108319), TrkB; #4603S), p-TrkB (#4619S), p-CREB (#9198S), CREB (#9197S), and β-actin (#4970S) at a 1:1000 dilution overnight at 4 °C. Anti-BDNF antibody was bought from Abcam (Cambridge, UK), and other primary antibodies were bought from Cell Signaling Technology Japan, K.K. (Tokyo, Japan). This was followed by incubation with horseradish peroxidase-conjugated secondary antibodies (#7074S, 1:1000, Cell Signaling Technology Japan) for 1 h. Immunoreactive bands were detected using an enhanced chemiluminescence detection kit, and a Light Capture AE-6971/2 device (ATTO Corp., Tokyo, Japan) was used for visualization. Band intensities were normalized to β-actin, total TrkB, or CREB using CS Analyzer 4 (ATTO Corp.).
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4

Non-reducing SDS-PAGE Protein Analysis

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Non-reducing SDS-PAGE at 12% acrylamide running gel with a 4% acrylamide
stacking gel on a Mini-Protean Tetra Cell (Bio-Rad) and Power pack
Basic (Bio-Rad) was performed following standard lab procedures. Precision
Plus Protein Dual Xtra Prestained Protein Standards molecular weight
marker (Bio-Rad) was co-run to estimate protein weights. Samples (15
μL) were mixed with 2× Laemmli Sample Buffer (Bio-Rad)
(15 μL) and 30 μL of each mixture was loaded in the wells.
All gels were run with a running buffer solution [25 mM Tris, 192
mM glycine, 0.1% (w/v) SDS] under a constant current of 30 mA until
bromophenol blue reached the bottom of the running gel. The gels were
stained using Bullet CBB Stain One(Ready To Use) (Nacalai Tesque,
Inc.) at room temperature for 15 min. Gels were imaged using WSE-5300
Printgraph CMOS I (ATTO Corp.) without de-staining. Densitometric
evaluations were performed using the image analysis software CS Analyzer
4 (ATTO Corp.).
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5

Hippocampal Protein Expression and Phosphorylation Analysis via Western Blotting

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Western Blotting for analysis hippocampal protein expression and phosphorylation was conducted as our previous report16 (link). Homogenized hippocampal lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to Amersham Hybond P PVDF 0.45 membranes (Bio-Rad, Hercules, CA, USACytiva, Tokyo, Japan). Membranes were incubated with 5% bovine serum albumin (BSA) in TBST for 1 h, followed by incubation with primary antibodies against BDNF (ab108319), TrkB; #4603S), pTrkB (#4619S), pCREB (#9198S), CREB (#9197S), and β-actin (#4970S) at a 1:1000 dilution overnight at 4 °C. Anti-BDNF antibody was bought from Abcam (Cambridge, UK), and other primary antibodies were purchased from Cell Signaling Technology Japan, K.K. (Tokyo, Japan). This was followed by incubation with horseradish peroxidase-conjugated secondary antibodies (#7074S, 1:1000, Cell Signaling Technology Japan) for 1 h. Immunoreactive bands were detected using an enhanced chemiluminescence detection kit, and a Light Capture AE-6971/2 device (ATTO Corp., Tokyo, Japan) was used for visualization. Band intensities were normalized to β-actin, total TrkB, or CREB using CS Analyzer 4 (ATTO Corp.).
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6

N-Homocysteinylation of HDL Characterization

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N-homocysteinylation of HDL was performed according to a previously reported method (Ishimine et al. 2010) . HDL isolated from pooled healthy serum was incubated with 0, 1, or 10 mM HcyT (MP Biomedicals, California, USA) at 37 °C for 24 h. Then, the treated samples were dialyzed against with 3 × 2 l PBS by using Cellulose Tubing (20/32) (Viskase Companies Inc, Illinois, USA). HcyT-treated HDL (50 μl at 2 mg protein/ml in PBS) were mixed with a 2-aminoethanethiol hydrochloride solution (dissolved in 20 mM ] to adjust the pH to near neutral) at a ratio of HDL protein: 2-aminoethanethiol 1:6.7 (w/w) and incubated at 37 °C for 12 h. To confirm the production of N-Hcy apoA-I in HDL, 2-aminoethanethioltreated samples were isolated by isoelectric focusing (IEF) in a pH range from 4.0 to 6.5 and transferred onto PVDF membranes, which were then incubated with anti-apoA-I antibody. Then, the membranes were incubated with HRP-conjugated rabbit anti-goat IgG. The antibody incubations were performed at room temperature for 1 h. The bands containing apoA-I were visualized with 3,3′-diaminobenzidine tetrahydrochloride and H 2 O 2 . The relative amount of N-Hcy apoA-I were analyzed with CS Analyzer4 software (ATTO CORPORATION). To calculate the ratio of N-Hcy apoA-I to total apoA-I, we defined N-Hcy apoA-I bands by a higher positive charge than intact apoA-I.
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7

Quantification of Serum Amyloid A in HDL

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HDL was isolated from plasma samples from patients with varying troponin I levels and healthy human plasma. The isolated HDL was analyzed by SDS-PAGE with CBB-R250 staining. Single band at the 14 kDa position was regarded as serum amyloid A (SAA) and the relative amount of SAA to total HDL protein was quantified with CS Analyzer4 software (Atto Corporation).
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8

Automated Western Blot Analysis

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Total proteins of the cells were isolated by using radio immunoprecipitation assay (RIPA) buffer (ATTO Corporation), and the protein concentration was measured using bicinchoninic acid (Sigma-Aldrich) assay. Immunoblotting was conducted on a JESSTM Simple Western automated nano-immunoassay system (ProteinSimple, USA). A mixture of proteins, fluorescent 5× master mix, 400 mM dithiothreitol (ProteinSimple) and biotinylated molecular weight markers was prepared, and then denatured at 95°C for 5 min. Primary antibodies were diluted with antibody diluent. Table 2 shows the information of the primary antibodies used in this study. The working solutions of HRP-conjugated anti-mouse or rabbit secondary antibodies included in this kit were used. For chemiluminescence detection, luminol-peroxide mix was prepared. Assay protocol was as follows: 25 min for separation time, 20 min for blocking, 45 min for primary antibody reaction, 45 min for secondary antibody reaction. The Compass Simple Western software (ver. 6.0.0; ProteinSimple) was used to obtain the chemiluminescent band images. The band intensities were quantified with CSAnalyzer4 software (ATTO Corporation). GAPDH and its corresponding target proteins were analyzed from the same protein lysate. All protein expression levels were normalized to the GAPDH expression in each band.
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9

Western Blot Analysis of ApoA-I and ApoA-II

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Western blot analysis was performed as described previously (Kameda et al. 2012) . Briefly, proteins were separated by SDS-PAGE using 14% polyacrylamide gels under reducing conditions and transferred to PVDF membranes (Millipore, Massachusetts, USA). The membranes were incubated with goat anti-apoA-I or anti-apoA-II polyclonal antibodies, followed by incubation with HRP-conjugated rabbit anti-goat IgG. The antibody incubations were performed at room temperature for 1 h. Then, the bands containing apoA-I and apoA-II were visualized with 3,3′-diaminobenzidine tetrahydrochloride and H 2 O 2 . The relative amounts of apoA-I/apoA-II heterodimers were semi-quantified with CS Analyzer4 software (ATTO CORPORATION, Tokyo, Japan).
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