Chemidoc imager

Brains and spleens were harvested from the subset of mice that were not used for c-Fos and CRF immunohistochemistry, then flash-frozen in liquid nitrogen and stored at −80 °C. Tissues were weighed and homogenized with 8 times their weight of RIPA buffer plus protease inhibitors. Western blotting was performed with primary antibodies against tumor necrosis factor (TNF)-α and IL-1β (Santa Cruz, Dallas, TX, USA; 1:250), and secondary antibodies conjugated to HRP (Rockland, Limerick, PA, USA; 1:5000) as previously described [28 (link)]. TNF-α was quantified at both the 26 and 51 kDa bands to gauge relative amounts of the transmembrane and active trimeric isoforms [32 (link)], respectively. Blots were imaged on a ChemiDoc imager (BioRad, Hercules, CA, USA) and analyzed using ImageLab software (BioRad, Hercules, CA, USA), with the bands of interest normalized to total lane protein using the stain-free quantification method, as previously reported [28 (link)].

Cells were washed on ice with PBS and lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Na-deoxycholate, 2 mM EDTA, 1% NP-40, and 50 mM NaF) with protease inhibitors containing 1 mM benzamidine, 1 mM PMSF, and cOmplete Mini-EDTA-free protease inhibitors (Merck KGaA, Darmstadt, Germany). Cells were homogenized by sonication (three times, 1 s with 15 s breaks on ice) and cell lysates were cleared by centrifugation (10 min, 16,000 rpm at 4 °C). Protein (30 µg) was mixed with Laemmli buffer (60 mM Tris-Cl pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue, and freshly added 140 mM DTT) and boiled (5 min at 95 °C). Each sample was separated by SDS-PAGE, transferred onto 0.45 µm nitrocellulose membranes (GE Healthcare Life Sciences, Chicago, IL, USA). Blots were immunodetected with antibodies listed in Table 2. Bands signals were enhanced using a chemiluminescent substrate (Santa Cruz Biotechnology, Dallas, TX, USA) and captured by ChemiDoc Imager (Bio-Rad Laboratories, Hercules, CA, USA). Densitometry was performed using Image Lab.v.6.0.1 software (Bio-Rad Laboratories, Hercules, CA, USA).

Four 4 × stock solutions were prepared containing (i) Ube1 (R&D Systems) + UbcH5c/UBE2D3 (R&D Systems) (400 nM Ube1 and 4 μM UbcH5c), (ii) Ubiquitin (R&D Systems) (1 mM Ub), (iii) CHIP + substrate + DnaJB4 (4 μM CHIP, 4 μM substrate, varying concentrations of DnaJB4), and (iv) ATP + MgCl₂ (10 mM ATP and 10 mM MgCl₂) in Ubiquitination assay buffer (50 mM Tris pH 8.0, 15 mM NaCl). CHIP + substrate + DnaJB4 solutions were allowed to equilibrate at room temperature for 30 min prior to initiating the reaction. Ubiquitination reactions were generated by adding 10 μl of each 4 × stock, in order from 1 to 4, for a final volume of 40 μl(100 nM Ube1, 1 μM UbcH5c, 250 μM Ubiquitin, 2.5 mM ATP, 2.5 mM MgCl₂, 1 μM CHIP and 1 μM substrate). Reactions were incubated at room temperature (10 min for chaperone Ubiquitination, 30 min for tau Ubiquitination), quenched in 20 μl 3 × SDS-PAGE loading buffer (188 mM Tris-HCl pH 6.8, 3% SDS, 30% glycerol, 0.01% bromophenol blue, 15% β-mercaptoethanol), and heated to 95 °C for 5 min. Samples were separated by SDS-PAGE on 4% to 20% polyacrylamide gels (Bio-Rad) and analyzed by in-gel fluorescence on a Chemidoc Imager (Bio-Rad) using the SYBR green fluorescence setting or by staining with Coomassie blue reagent. Quantitation of substrate Ubiquitination was performed by densitometry analysis in ImageJ (NIH), which was thresholded to a no-CHIP control.

Proteins (3 μg per lane) mixed with NuPage LDS sample buffer (1X, Thermo Scientific, Waltham, MA, USA cat # NP0008) were separated by electrophoresis in pre-cast 12% Bis Tris NuPage gels (1.0 mm × 12 well, Thermo Scientific, Waltham, MA, USA cat. # NP0342BOX). HLA-DQ and CD63 were identified by Western blotting using antibodies indicated below and goat anti-mouse or human HRP secondary antibodies (1 in 10,000 in TBS-T) and a chemiluminescence detection kit (Thermo Scientific, Waltham, MA, USA, cat # 89880). Images were obtained using a ChemiDoc imager (Bio-Rad, Hertfordshire, United Kingdom). Nytran filters were stained in Ponceau S solution (Thermo Scientific, Waltham, MA, USA, cat # ab270042) to enable normalization using common non-specific banding.

Proteins were separated using 12 or 4–15% Tris-glycine gels (Bio-Rad, Hercules, CA) and transferred to PVDF membrane (Millipore) using semi-dry transfer (Bio-Rad). The blots were incubated for 1 h at room temperature in 5% (w/v) non-fat dry milk (Bio-Rad) in TBST buffer (10 mM Tris pH 7.5, 150 mM NaCl, 0.1% Tween20) followed by the overnight incubation at 4°C with primary antibody. After 1 h incubation at room temperature with a 1:10,000 dilution of HRP-conjugated anti-mouse or anti-rabbit IgG F(ab)₂ (GE Healthcare Life Sciences, Piscataway, NJ), blots were developed using ECL Plus Western Blotting Detection Reagents (GE Healthcare Life Sciences, Piscataway, NJ) (3 (link)). The protein bands were quantitated with ChemiDoc Imager and Image Lab^TM software (Bio-Rad, Hercules, CA).