Anti total iκbα

Three hundred microliters of intact-harvested cells were pelleted at 400×g for 5 min and resuspended in 50 μL of 1× Laemmli SDS-PAGE buffer (0.1 % 2-mercaptoethanol, 0.0005 % bromophenol blue, 10 % glycerol, 2 % SDS, 2 %, 63 mM Tris-HCl pH 6.8) and heated to 100 °C for 2 min. Proteins were separated on polyacrylamide gels (Pierce Biotechnology, Rockford, IL, USA), transferred to nitrocellulose membranes, blocked, and probed using anti-total IκBα (Cell Signaling Technology) and secondary-HRP (Sigma-Aldrich Co.) antibodies in 5 % (w/v) milk powder-PBS. Membranes were soaked in chemiluminescent substrate (Pierce Biotechnology) and imaged.
For IκBα ELISA, cells were scraped into 1 mL of ice-cold PBS, pelleted at 500×g for 5 m and resuspended in 100 μL of 1 % Triton (v/v) in PBS with protease inhibitors (Pierce protease inhibitor mini tablets, EDTA-free; Fisher Scientific Ireland Ltd, Dublin, Ireland). A BCA protein assay was performed and equal quantities of protein added to a PathScan® Total IκBα Sandwich ELISA (Cell Signaling Technology, Danvers, MA, USA) as per manufacturer’s instructions. Total IκBα present was expressed as fold of the control no-stretch group.

Amaxa^® Cell Line Nucleofector^® Kit T and Amaxa^® Glia Cell Nucleofector^® Kit T were purchased from LONZA. Primers for quantitative RT-PCR were synthesized by Life Technologies. SYBR Green for quantitative RT-PCR was purchased from Roche. SP600125, Bay11–7082, SB203580, U0126 and LPS were purchased from Sigma. Amyloid-β 42 (Aβ42) peptide was purchased from AnaSpec. Oligomeric Aβ42 was prepared as previously described (Huang et al., 2015 (link)). Antibodies used in this study are as followed: anti-phospho-p38-MAPK, anti-total-p38-MAPK, anti-phospho-ERK1/2, anti-total-ERK1/2, anti-phospho-JNK, anti-total-JNK, anti-phospho-IκBα, anti-total-IκBα, anti-phosho-NF-κB, anti-total-NF-κB, anti-phospho-c-Jun, anti-total-c-Jun and anti-β-actin were purchased from Cell Signaling Technology; anti-tubulin (Millipore); anti-mouse IgG and anti-rabbit IgG antibody conjugated with horseradish peroxidase (ThermoFisher Scientific).