Figure S1. Phosphorylation of �� and �� isoforms of GSK3 (at serine 21 for the �� isoform and serine 9 for the �� isoform) are both increased by treating SH-SY5Y cells with PBT2. Cells were treated for 1?hr in serum-free media (which contains 1.5?��M Zn and 5.2?nM Cu) before analyzing cell extracts by Western blot for levels of phosphorylated GSK3��/�� (pGSK3��/��). Data are mean values (��SEM) expressed relative to phosphorylated GSK3��/�� (pGSK3��/��) in cells treated with 0?��M PBT2 and represent densitometry analysis of Western blots MYO10
as shown in Fig. 1A. n?=?3 replicate Western bots. Figure S2. Treating SH-SY5Y cells with PBT2 decreases Cu content of the culture media and increases cellular Cu in cells. Cells were treated in serum-free DMEM:F12 media for 1?hr as per Fig. 1. Cu content of media and pelleted cells was determined by ICP-MS. Data are expressed relative to Cu levels in control treated cells (�� standard error, n?=?3). **Denotes values significantly different (p?<?0.01) <a href="http://www.selleckchem.com/products/voxtalisib-xl765-sar245409.html
compared to control treated cells (two tail t-test). Figure S3. Zn- and Cu-dependent phosphorylation of GSK3 in SH-SY5Y cells treated with PBT2. (A) Cells were treated for 1?hr in metal ion free HBSS before analyzing cell extracts by Western blot for levels of pGSK3��/��, total GSK3��, and the control protein ��-actin. The HBSS was supplemented with 10?��M PBT2 and 10?��M Zn or 10?��M Cu as indicated before adding to the cells. (B) Cells were treated as per (A) except the media was supplemented with Fe (as FeCl2), Al (as Al2(SO4)3) or Li (as LiCl). All Western blot data are representative of separate replicates (n?=?3). Figure S4. Phospho-protein kinase A (pPKA-C), phospho-protein kinase C (pPKC) and casein kinase-1 (CK1) levels in SH-SY5Y cells treated with PBT2+Zn. Cells were treated for 1?hr in metal ion free HBSS before analyzing cell extracts by Western blot. Where indicated the HBSS was supplemented with 10?��M PBT2 and 10?��M Zn before adding to the cells. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized Peptide 17 order
for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. ""J. Neurochem. (2010) 115, 1495�C1507. Endothelial tight junctions and efflux transporters of the blood�Cbrain barrier (BBB) significantly limit brain accumulation of many drugs, including protease inhibitors such as saquinavir. The cholinergic agonist nicotine is one of the most commonly used drugs in the world and the incidence is even higher in the human immune deficiency virus population (?70%). We examined the ability of nicotine and its primary metabolite cotinine to modify brain uptake of saquinavir in rats. Both nicotine and cotinine at pharmacological concentrations matching those in smokers, increased brain saquinavir uptake by two fold.