Fuji medical x ray films

Two week old cortical neurons cultured in either 60 mm or 6-well plates were treated by resveratrol, AICAR or other chemicals as stated. For blockade/occlusion experiments, unless specifically stated, the cells were pre-treated for one hour prior to resveratrol treatment then remained in the bath until cell lysis. Cells in control groups were treated with the appropriate vehicle solvents (saline or DMSO). After treatment, neurons were lysed in Laemmli 2X sample buffer (4% SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, 0.125 M Tris HCl) and boiled for 10-min at 95°C for SDS page electrophoresis. After separation in SDS page, proteins were transferred to PVDF immunoblotting membrane (Bio-rad) and probed for different targets with the stated antibodies. Immunoblots were visualized using a chemiluminescence detection system (GE Healthcare) and exposed to Fuji medical X-ray films (Fisher Scientific), scanned and analyzed using the NIH ImageJ program.

Human hippocampal tissues from control and AD patients were provided by National Institutes of Health (NIH) NeuroBioBank.
Transfected HEK cells or rat cultured cortical neurons used for western analysis were first rinsed with cold PBS and scraped from the culture dishes with RIPA lysis buffer [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate (SDOC) and 1% sodium dodecyl sulfate (SDS)] containing protease inhibitors (Roche). After sonication, the lysates were centrifuged for 10 min at 13,000 rpm.
For the ubiquitination assay, the supernatant of 500 μl in RIPA buffer was incubated with antibodies against GluA1 and protein A-Sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 15 h at 4°C. Immunocomplexes were washed three times with ice-cold NP-40 buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 1% SDOC), resuspended in 2X Laemmli buffer, and denatured at 95°C for 10 min.
Prepared cell lysates or tissue lysates were subject to SDS-PAGE. Proteins were transferred to PVDF immunoblotting membranes (Bio-Rad) and probed for different targets with the stated antibodies. Immunoblots were visualized using a chemiluminescence detection system (GE Healthcare) and exposed to Fuji medical X-ray films (Fisher Scientific), scanned, and analyzed using the NIH ImageJ program.