Anti hsp70

Cells were transfected with Lipofectamine 2000 (Invitrogen), according to the manufacturer's instructions. Briefly, cells were plated at 20%–30% density in 12‐well plates 24 h prior to transfection. For siRNA transfection, the equivalent of 200 nM of siRNA per well of a 12‐well plate was utilized. After a 48 h incubation period, the lysates of the cells were measured using the Bradford assay. Equal amounts of protein were separated by SDS‐PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membranes were immunoblotted with the following specific antibodies: anti‐HSP70, anti‐rabbit IgG‐HRP (Sigma‐Aldrich), anti‐rat IgG‐HRP (Sigma‐Aldrich), and anti‐GAPDH (Sigma‐Aldrich), using standard protocols. The blots were developed with West Dura chemiluminescent substrates using a Bio‐Rad ChemiDoc imaging system.

PKC epsilon, SOD2, Nrf-2, and Myogenin protein content was tested with a Western blot. In brief, C2C12 was lysed with a RIPA buffer and total protein was quantified with a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the instruction protocol. Forty micrograms of proteins were separated on 10% SDS-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and incubated with a specific primary antibody diluted in the blotting solution as recommended by the manufacturer protocol. We used anti-PKCe (Merck Millipore, Darmstadt, Germany, Cat. No.: 06-991), anti-SOD2 (Enzo Life Sciences Inc., Farmingdale, NY, USA, Cat. No.: ADI-SOD-111F), anti-Myogenin (Santa Cruz, Dallas, TE, USA, Cat. No.: sc-12732), anti-Nrf2 (clone: D179C, Cell Signaling, Danvers, MA, USA, Cat. No.: 12721), and anti-HSP70 (Sigma-Aldrich, St. Louis, MO, USA, Cat. No.: H5147) as a loading control. The nitrocellulose membranes were then washed and incubated with 1:5000 peroxidase-conjugated anti-rabbit (Thermo Fisher Scientific, Waltham, MA, USA) or 1:2000 peroxidase-conjugated anti-mouse IgG (Sigma-Aldrich, St. Louis, MO, USA). Proteins were resolved with an ECL SuperSignal West Pico Chemiluminescent Substrate Detection System (Thermo Fisher Scientific, Waltham, MA, USA) and densitometric analyses were performed using ImageJ software (by NIH).

Western blot assays with fly heads were performed as previously described (Garbe et al., 2013 (link)). Briefly, 7–10 fly heads per genotype/condition were lysed in 1x Passive Lysis Buffer (Promega), supplemented with protease and phosphatase inhibitors. For S2 cell extracts, 48 hr after transfection, cells were collected and lysed in the same buffer as described for fly heads. The following primary antibodies were used: anti-PER (UP1140, 1:1000), anti-TIM (UPR42, 1:1000) and anti-HSP70 (Sigma, 1:5000).

Both primary and BV-2 microglial cells, control and treated, were washed with 1× phosphate-buffered saline (PBS) thrice and were fixed with acetone to methanol followed by permeabilization with 0.3 % Triton X-100 in PBS (PBST). Cells were then incubated with anti-α-tubulin (1:500) or anti-NF-kB (1:300) or anti-AP1 (1:300) or anti-HSP-70 (1:300) (from Sigma-Aldrich) diluted in 2 % BSA, for 24 h at 4 °C in humid chamber. After two to three washings with 0.1 % PBST, the cells were incubated with the secondary antibody anti-mouse IgG 488 and anti-rabbit IgG 488 (prepared in 2 % BSA (1:500)) for 2 h at room temperature. Cells were incubated with (DAPI, 1:5000 in 1× PBS) for 10 min for nuclear staining and then mounted with anti-fading reagent (Fluoromount, Sigma). For CellRox and Mitotracker staining, after treatment period, the dye was added in culture according to the manufacturer’s instructions; cells were then fixed and mounted. Images were captured using Nikon AIR Confocal Laser Microscope and analyzed using NIS elements AR analysis software version 4.11.00. Experiment was performed in triplicate.

Liver, spleen, and total brain were harvested at indicated time points, immediately frozen in liquid nitrogen, and stored at −80 °C. Tissues were lysed using 1× RIPA buffer (9806, Cell Signaling) supplemented with proteases (Complete Mini protease inhibitor, Roche, Almere, The Netherlands) and phosphatase inhibitors (PhosSTOP, Roche, Almere, The Netherlands). Protein concentration was determined by the Bradford method and 30 µg total protein extract was loaded, separated by 10% SDS Page gel, blotted onto nitrocellulose membrane that was blocked with 5% milk in PBST for 2 h to prevent a-specific binding. Bvra was detected using the anti-Bvra primary antibody (1:1000, ADI-OSA-450, Enzo, Pero, MI, Italy) incubated overnight in 5% BSA, PBST. Anti-actin (Sigma-Aldrich, Milano, Italy) antibody or Anti-HSP70 (ADI-SPA-185D) were used as loading controls.