peutic molecules with disease modifying activities, which are urgently needed to tackle the ill effects of a highly complex, multi factorial disease like AD. Experimental Section Test Compounds Withanolide Bafetinib INNO-406 A and asiatic acid were purchased from Chromadex Incorporation. The purity of 1 and 2 was 99.3% and 93.7%, respectively. Isolation and Culture of Primary Rat Cortical Neurons Primary cortical neurons were isolated from 1 day old Sprague Dawley rat pups and cultured as described by Chandler et al.58 All procedures were performed according to guidelines developed by the Institutional Animal Care and Use Committee at Michigan State University. The cells were plated on poly D lysine coated, 12 well plates at a concentration of 1 × 106 cells per well in fresh cortical medium.
The experiments were performed on 3 4 day old neuronal cultures. The cells were treated with 1 and 2 at different doses for 24 h. Immunostaining of Primary β-Sitosterol 83-46-5 Rat Cortical Neurons To perform the immunofluorescence microscopic study, neuronal cultures were fixed for 20 min in 4% paraformaldehyde and then incubated for 20 min in blocking solution. After washing 2X with PBS, cells were labeled overnight at 4 with primary antibody for neurons. After 3X PBS washes, primary antibodies were detected with rhodamine conjugated secondary antibody. The cells were visualized with an inverted fluorescence microscope Leica DM IL using a 40X objective lens. Western Blot Analysis The following antibodies used for western blotting: anti BACE1, anti ADAM10, anti IDE, anti NEP, anti APP, C terminal, anti APP, N terminal and Actin.
To detect secreted protein, conditioned media were collected and processed as explained earlier.59 To detect cellular proteins, cells were washed three times with ice cold TBS and lysed for 30 min by scraping into ice cold radioimmunoprecipitation assay buffer.60 The total cell lysate was obtained by centrifugation at 12,000 rpm for 30 min at 4. The total protein concentration was measured by using a BCA protein assay kit from Pierce. Equal amounts of total protein from each condition were run at 200 V on 10% Tris HCl gels, 5% gels, and 10 20% Tris Tricine gels. The separated proteins were transferred to polyvinylidene fluoride membranes and nitrocellulose membranes for 1 h at 100 V and incubated at 4 overnight with the appropriate primary antibodies.
Blots were washed three times in PBS Tween and incubated with appropriate HRPPatil et al. Page 7 J Nat Prod. Author manuscript, available in PMC 2011 July 23. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript linked secondary antibodies diluted in PBS T for 1 h at room temperature. After washing three times in PBS T, blots were developed with the Pierce SuperSignal West Femto Maximum Sensitivity Substrate and imaged with the BioRad ChemiDoc. Quantity One software from Bio Rad was utilized to quantify the signal intensities of the protein bands. Statistical Analysis Data are shown as means S.D. for the indicated number of experiments. The Student,s ttest was used to evaluate statistical significances between different treatment groups. Statistical significance was set at p 0.05.
Supplementary Material Refer to Web version on PubMed Central for supplementary material. Patil et al. Page 8 J Nat Prod. Author manuscript, available in PMC 2011 July 23. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript Acknowledgments We thank L. Liu, N. Tran and H. Geekiyanage for isolating primary rat neuronal cells, and A. Abramczyk for preparing SDS PAGE gels. We also thank A. Rillorta from Chromadex, Inc. for affording pure test compounds in timely manner. This work was funded in part by the MSU Foundation, the National Institutes of Health, the National Science Foundation, and t