For proteomic analysis, differentiated SH-SY5Y cells were treated with plain and hill extracts (10 μg/mL) for 24 h as described above. At the end of treatments, cells were collected and washed with PBS. After centrifugation (1000× g for 5 min), the resulting pellets were immediately frozen and stored at −80 °C until use. For proteomic studies, each condition was performed in triplicate.
Cell pellets were resuspended in rehydration solution [44 (link)] and protein contents of resulting protein extracts were measured with an RC-DC Protein Assay from Bio-Rad.
The 2DE was carried out as previously described [45 (link)]. Briefly, 200 µg of proteins were filled up to 450 μL in rehydration solution. Immobiline Dry-Strips (GE Health Care Europe; Uppsala, Sweden); 18 cm, nonlinear gradient (pH 3–10) were rehydrated overnight in the sample and then transferred to the Ettan IPGphor Cup Loading Manifold (GE Healthcare) for isoelectrofocusing (IEF). The second dimension (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis; SDS-PAGE) was carried out by transferring the proteins to 12% polyacrylamide, running at 16 mA per gel and 10 °C for about 16 h, using the Protean® Plus Dodeca Cell (Bio-Rad). The gels were stained with Ruthenium II tris (bathophenanthroline disulfonate) tetrasodium salt (Cyanagen Srl, Bologna, Italy) (RuBP). ImageQuant LAS4010 (GE Health Care) was used for the acquisition of images. The analysis of images was performed using Same Spot (v4.1, TotalLab; Newcastle Upon Tyne, UK) software. The spot volume ratios among the three different conditions (Control, hill, plain) were calculated using the average spot normalized volume of the three biological replicates performed in duplicate. The software included statistical analysis calculations.
Cell pellets were resuspended in rehydration solution [44 (link)] and protein contents of resulting protein extracts were measured with an RC-DC Protein Assay from Bio-Rad.
The 2DE was carried out as previously described [45 (link)]. Briefly, 200 µg of proteins were filled up to 450 μL in rehydration solution. Immobiline Dry-Strips (GE Health Care Europe; Uppsala, Sweden); 18 cm, nonlinear gradient (pH 3–10) were rehydrated overnight in the sample and then transferred to the Ettan IPGphor Cup Loading Manifold (GE Healthcare) for isoelectrofocusing (IEF). The second dimension (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis; SDS-PAGE) was carried out by transferring the proteins to 12% polyacrylamide, running at 16 mA per gel and 10 °C for about 16 h, using the Protean® Plus Dodeca Cell (Bio-Rad). The gels were stained with Ruthenium II tris (bathophenanthroline disulfonate) tetrasodium salt (Cyanagen Srl, Bologna, Italy) (RuBP). ImageQuant LAS4010 (GE Health Care) was used for the acquisition of images. The analysis of images was performed using Same Spot (v4.1, TotalLab; Newcastle Upon Tyne, UK) software. The spot volume ratios among the three different conditions (Control, hill, plain) were calculated using the average spot normalized volume of the three biological replicates performed in duplicate. The software included statistical analysis calculations.
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