Mouse anti hsp90

Proteins were extracted from 14–17 DIV neuronal culture using an extraction buffer: 150 mM NaCl, 50 mM Tris-Cl (pH 8.0), 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS with a complete mini, EDTA-free protease inhibitors cocktail (Roche Applied Science). The extracts were quantified using the BCA Protein assay kit (QuantumProtein Assay, EuroClone) as per manufacturer instruction. The protein suspension was dissolved in reducing Laemli sample buffer and heated for 5 min at 50 °C. Thirty micrograms of the protein extract was loaded per lane and separated by SDS-PAGE (12.5% polyacrylamide gel) and subsequently transferred onto nitrocellulose membrane (GE Healthcare #10600001) and probed using the following antibodies: MitoProfile total OXPHOS rodent WB antibody cocktail (Abcam ab110413) 0.6 µg/mL; mouse anti Hsp90 1:1000 (BD Bioscience #610418). Species-specific, HRP-conjugated secondary antibodies (BioRad) have been used. Immuno-bands were visualized using the chemiluminescence reagent Westar Sun (Cyanogen) on an UviTech Mini HD9 system (Eppendorf). Band intensities were analyzed using the image processing software ImageJ (NIH, USA), using the HSP90 signal for normalization.

Total cell protein was isolated from pelleted neutrophils using cell lysis buffer. Absolute protein content of lysates was determined by Bradford assay (Bio-Rad, Hercules, CA, USA). Samples were boiled at 95°C for 5 min and then were run on 12% SDS-PAGE gels. Proteins were transferred onto nitrocellulose membranes, blocked with 5% fat-free milk in TBST for 1 h, and detected using rabbit anti-MLKL antibody-N-terminal (Abcam), mouse anti-caspase-8 (Enzo Life Sciences), and mouse anti-Hsp90 (BD Biosciences) monoclonal primary antibodies. Anti-rabbit MLKL, anti-mouse caspase-8, and anti-mouse-Hsp90 secondary antibodies (all from Abcam) were then applied to membrane, which were subsequently incubated with Western Blotting Detection Reagent (Thermo Scientific) and imaged using ImageQuant LAS 4000 System (GE Healthcare).

Proteins were extracted using RIPA lysis buffer (15 mM Tris-HCl, 1%, Triton X-100, 0.1%, SDS, 167 mM NaCl, 0.5% sodium deoxycholatic acid), with protease inhibitors cocktail (Sigma-Aldrich, Rehovot, Israel). Total protein (30 μg) was loaded. SDS/PAGE, protein transfer and Western blotting were performed using standard laboratory techniques [36 ]. Primary antibodies were: goat anti–XBP1 (1–2 µg/ml, Abcam, Zotal, Tel-Aviv, Israel), mouse anti-Hsp90 (1:3000, BD Biosciences), mouse anti-ATF6 (1–5 µg/ml Abcam, Zotal, Tel-Aviv, Israel), rabbit anti- TNF–α (0.1 - 0.2 µg/ml, Abcam, Zotal, Tel-Aviv, Israel), mouse anti-IL6 (0.2–0.4 µg/ml, Abcam, Zotal, Tel-Aviv, Israel), rabbit anti- TGFβ (0.1–1 µg/ml, Abcam, Zotal, Tel-Aviv, Israel), goat anti PL (1:100–1000, Santa Cruz Biotechnology, Almog, Shoham, Israel). The following secondary antibodies were used: goat anti mouse IgG (1:5000, Millipore, Mercury, Rosh Haayin, Israel) and goat anti rabbit IgG (1:3000 Abcam, Zotal, Tel-Aviv, Israel). Luminata-crescendo western HRP substrate chemiluminescence (Millipor, Mercury, Rosh Haayin, Israel) was used to develop the blots. Densitometry analyses of immunoblots were performed using imageQuantTL software (GE life sciences, NJ, USA). All proteins were quantified relative to house-keeping gene.