N which had been calibrated with 50 mM Tris/HCl, pH 8.0, 1 mM

N which had been calibrated with 50 mM Tris/HCl, pH 8.0, 1 mM EDTA. The protein was eluted with 1 M NaCl and the eluate dialyzed and analyzed by SDS-PAGE. For expression in AX2 the RACK1 cDNA was cloned into pBsr-N2-GFP vector (N-terminal GFP) and expressed as GFP-RACK1 under control of the actin 15 promoter and also into mRFPmars plasmid (N-terminal RFP) for RFP-RACK1 [82,83]. A PCR-mediated sitedirected mutagenesis (QuikChange Site-Directed Mutagenesis Kit, Stratagene) was used to generate mutations in the Methyl 3-amino-2,4-dichloro-5-fluorobenzoate GST-RACK1 and GFP-RACK1 plasmids. The mutations were confirmed by sequencing.Phosphoinositide binding assayFor expression of recombinant D. discoideum RACK1 as glutathione S transferase (GST) fusion protein in E. coli, a full-length cDNA was cloned into pGEX-4 T-1 vector (GE Healthcare Life Sciences). E. coli strain XL1 Blue was used for expression of the GST fusion protein. Induction of protein expression was with 0.25 mM isopropyl -D-thio-galactoside (IPTG) when an OD600 of 0.8 was reached. Cells were further cultured at 30 for 3 hours. They were harvested, lysed in 50 mM Tris/HCl, pH 7.4 to 8.0, 100 mM NaCl, supplemented with Protease inhibitors (0.5 mM PMSF, 1 mM Benzamidine and Complete (Roche) and 1 mM DTT) with an EmulsiFlex cell homogenizer (Avestin Europe GmbH, Mannheim,PIP-strips supplied by Echelon Biosciences, Inc. (Salt Lake City, Utah, USA) were used to perform phosphoinositide binding according to the supplied protocol. Briefly, GST and GST-fusion proteins were eluted from the glutathione agarose beads with elution buffer (20 mM reduced glutathione, 50 mM Tris/HCl, pH 7.4, 100 mM NaCl, 0.2 Tween-20, and 100 mM DTT). The membranes were blocked with 0.1 ovalbumin (Sigma # A-5253) in TBS for one hour at room temperature. After discarding the blocking solution membranes were incubated with 1 mg/ml GST-fusion proteins in TBS-T tert-Butyl (2-bromothiazol-5-yl)carbamate (50 mM Tris/HCl, pH 7.4, 100 mM NaCl, 0.2 Tween-20) at room temperature for one hour. The protein solution was then discarded and the membranes were washed with TBS-T three times 10 minutes each. Bound protein was detected by western blot analysis with GST polyclonal antibodies as primary and anti-rabbit IgG-peroxidase (Sigma # A-6154) as secondary antibody followed by enhanced chemiluminescence.Lipid vesicle preparation and sedimentation assayPhosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), PI (3) P, PI (4) P, PI (5) P, PIOmosigho et al. Cell Communication and Signaling 2014, 12:37 http://www.biosignaling.com/content/12/1/Page 15 of(3,4) P2, PI (3,5) P2, PI (4,5) P2, and PI (3,4,5) P3 were obtained from Sigma and diluted in chloroform. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13485127 Liposome binding experiments were performed with a modified published liposome binding assay protocol [84]. Lipid mixtures containing 65 PC, 20 PE, 5 PS and 10 individual phosphoinositides were produced by mixing appropriate lipid solutions in chloroform/methanol. Slow flow nitrogen gas was used for the production of a film on the glass and vacuum desiccation for 30 min for solvent removal. Sterile-filtered sucrose binding buffer (20 mM HEPES, pH 7.4, 100 mM KCl, 1 mM EDTA, 0.1 M sucrose) was added to a final lipid concentration of 1 mg/ml and incubated at 37 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14445666 for 2 h. Lipids were then sonicated in a waterbath-sonicator for 10 sec. To test liposome binding, a 100 l reaction mixture of freshly prepared liposomes and 5 g of purified protein were incubated for 15 min at room temperature and centrifuged at 100,000 ?g (42,000 rpm) a.