Gs 700 densitometer

COX-2 and perilipin-2 protein levels in cultured macrophages were detected by Western blotting. Aliquots of MT-III-stimulated and non-stimulated cells (1.5×10⁶ cells) were lysed with 100 μL sample buffer (0.5 M Tris-HCl, pH 6.8, 20% SDS, 1% glycerol, 1 M β-mercapto ethanol, 0.1% bromophenol blue) and boiled for 10 min. Samples were resolved by SDS-PAGE on 10% bis-acrylamide gels overlaid with a 5% stacking gel. Proteins were then transferred to nitrocellulose membranes using a Mini Trans-Blot (Bio-Rad Laboratories, Richmond, CA, USA). The membranes were blocked for 1 h with 5% nonfat dry milk in Tris-buffered saline (TBS) (20 mM Tris, 100 mM NaCl, and 0.5% Tween 20, pH 7.2), and incubated with primary antibodies against COX-2 (1∶1000 dilution), perilipin-2 (1∶2000) and β-actin (1∶3000) for 1 h. They were then washed and incubated with the appropriate secondary antibody conjugated to horseradish peroxidase. The immunonereactive bands were detected by the entry-level peroxidase substrate for enhanced chemiluminescence (ECL), according to the instructions of the manufacturer (GE Healthcare). Band densities were quantified with a GS 700 densitometer (Bio- Rad Laboratories) using the image analysis software Molecular Analyst (Bio-Rad Laboratories).

Immunoblot (IB) experiments were performed as described previously (Jones et al., 2004 (link), 2007 (link); Balut et al., 2010a (link),b (link); Gao et al., 2010 (link); Bertuccio et al., 2014 (link); Farquhar et al., 2017 (link)). Briefly, cells were lysed and protein concentrations were determined by the BCA protein assay (Walker, 1994 (link)). Equal amounts of protein (30 μg) were loaded into wells of a gel (6 or 8%) and protein standard (8 μl) used (BenchMark™ pre-stained protein ladder; Invitrogen, Cat No. 10748-010) and resolved with SDS-PAGE for 150 mV for 90 min (Hoefer Mighty Small II system, Cat. No. 80-6149-35, Amersham Biosciences Corp. Piscataway, NJ, USA). Proteins were transferred (50 V, 2 h) with a semi-dry transfer unit (Hoefer, EPS 2A200) to polyvinylidene difluoride (PVDF) membranes for further IB analysis with α-streptavidin antibody. Proteins bands were visualized by enhanced chemiluminescence detection (Lumilight, Roche, Basel Switzerland). Blots were probed for β-actin as a protein loading control. The bands obtained from immunoblot analysis were quantified by densitometry, using the GS-700 densitometer (Bio-Rad) and the Quantity One programme (BioRad laboratories). The obtained band intensities for the various time points were normalized to β-actin and then compared relative to the intensity at time 0 (t = 0) and reported.

Bax, Bcl-2, Bad, and Bcl-xl protein expression was determined on nuclear proteins separated from neuronal nuclei by SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes using a wet trans-blotting system (Ravishankar et al., 2001 (link)), as well as on cytosolic (Halat, 1992 (link)) and mitochondrial proteins (Lasso Pirot et al., 2007 (link)). The membranes were incubated with polyclonal anti-Bax, anti-Bcl2, anti-Bad, or anti–Bcl-xl antibodies (Santa Cruz Biotechnology). Immunoreactivity was then detected by incubation with horseradish peroxidase–conjugated secondary antibody (Rockland Immunochemicals). Specific complexes were detected by enhanced chemiluminescence method using the ECL detection system (Amersham Pharmacia Biotech) and analyzed by imaging densitometry (GS-700 densitometer, Bio-Rad). The densitometric scanning data were expressed as autoradiographic values per immunoblot protein. Concomitant actin protein gels were run to demonstrate the consistent amounts of protein in each sample. Samples were run in duplicates. Proteins were expressed as absorbance/OD (OD × mm²).