Dulbecco's modified eagle medium (DMEM), OptiMEM and gentamycine were obtained from Gibco (Eggenstein, Germany). Hoechst 33258, paraformaldehyde and α-tubulin antibody were from Sigma-Aldrich (Taufkirchen, Germany). Fetal bovine serum (FBS) and Trypsin-ethylenediaminetetraacetic acid (EDTA) for detaching SN56-cells were from PAA (Marburg, Germany). Horse radish peroxidase (HRP) conjugated antibodies were obtained from GE Healthcare (Freiburg, Germany).
Monoclonal PHP antibody
Monoclonal antibody of PHP was used as previously generated and characterized .
Cell culture and cell extracts
SN56.B5.G4 cells, a cholinergic murine neuroblastoma cell line, were provided by C. Culmsee (Marburg, Germany). The cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. For cultivation of SN56 cells DMEM supplemented with 10% FBS and gentamycine (50 μg/ml) was used. Experimental procedures with SN56 cells were performed with passages 25 - 40. At confluency, cells were split 1:3-1:5 by incubation with trypsin/EDTA-solution. After washing cells were cultivated in 75 cm2 flasks with 106 cells/flask.
Cell extracts for Western blot analysis were prepared by washing cells with ice-cold phosphate buffered saline (PBS; 13.7 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4) and were resuspended in homogenization buffer containing 50 mM Tris/HCl (pH 7.5), 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM benzamidine (BZ). For disruption cells were sonicated 3 times with 5 pulses (1/2 cycles and 60% amplitude; sonificator: Ultrason technology, Hielscher; Germany).
The total protein concentration was determined by using the Lowry assay  based on the protocol modified by Hartree .
A total amount of 50 μg protein and 100 ng recombinant (rec.) PHP were applied on 15% SDS-PAGE gels. Proteins were transferred onto nitrocellulose membrane by semidry blotting for 1 h at 10 V and then the membrane was blocked with 5% non-fat milk powder in TBST. Monoclonal primary PHP antibody (1:200, 0.1% bovine serum albumin), α-tubulin (1:10,000, 5% non-fat milk powder) as a loading control and the appropriate secondary antibodies conjugated to HRP(1:2,500, Sigma) were used to accomplish immunodetection of PHP and α-tubulin. Membranes were incubated with the primary antibodies over night at 4°C and with the secondary antibodies for 1 h at room temperature (RT). Immunoreactive bands were visualized by using enhanced chemiluminescence (Thermo Sientific, IL, USA) and automatic film processor Cawomat 200 IR (AGFA, Germany).
Transfection of SN56 cells
SN56 cells were washed with OptiMEM supplemented with 10% FBS and adjusted to 4,000,000 cells in 375 μl of the same medium. After adding 26 μg DNA or 25 μl of the siRNA solution (20 μM) in a 4 mm cuvette, electroporation was performed by using Genpulser Xcell (Bio-Rad; Munich, Germany). The cells from each transfection were seeded in a 6-well-plate for Western Blot analysis and ACh measurement. For Hoechst 33258 staining the transfected cells were seeded in 24-well-plates. Culture was achieved under standard conditions in DMEM containing 10% FBS, but no gentamycine.
Overexpression of PHP
Mammalian PHP overexpression vector was constructed by inserting polymerase chain reaction (PCR) generated cDNA, using php-pET-16b  as a template, in pIRES2-AcGFP1 (Takara Bio Europe/Clontech; Saint-Germain-en-Laye, France). The primer with the sequence 5'-atc gga att cca tgg cgg tgg cgg a-3' was used as a forward primer and with the sequence 5'-cgg atc cgt cag tag ccg tcg tta gc-3' as a reverse primer for amplifying the PHP sequence with PCR. Both, PHP and green fluorescent protein (GFP) were expressed from a single bicistronic mRNA.
Site-directed mutagenesis of histidine 53 of the human PHP sequence cloned in pIRES2-AcGF1-PHP was inserted by PCR using oligonucleotides (H53Asense: 5'-gca gaa gtg ggc tga gta cgc cgc gga cat cta cg-3', H53Aanti: 5'-cgt aga tgt ccg cgg cgt act cag ccc act tct gc-3') of the mutated residue. The overexpression vectors were transferred into SN56 cells by electroporation as described under transfection of SN56 cells.
Downregulation of PHP by siRNA
Transient downregulation of PHP was performed with chemically synthesized siRNA (Qiagen; Hilden, Germany). Several siRNA sequences were tested for their potency to downregulate PHP in SN56 cells. The siRNA corresponding to the nucleotides 5'-aac tga gaa gat caa agc caa-3' of the murine PHP sequence yielded best results. As negative control SN56 cells were transfected with scrambled siRNA. Transfection of SN56 cells with siRNA was performed as described in transfection of SN56 cells.
Analysis of cell damage
For nuclear staining with Hoechst 33258 cells were seeded on 24-well-plates and cultured under appropriate conditions. The cells were washed with 37°C PBS, fixed for 30 min with 4% paraformaldehyd and then incubated with Hoechst 33258 (10 μg/ml) for 30 min at RT. Nuclear morphology was analyzed with the fluorescence microscope Axiovert 25 from Zeiss (Oberkochen, Germany).
Sample preparation for ACh measurement was performed as described previously . In brief, SN56 cells were washed with PBS and were detached with trypsin-EDTA solution. Then trypsin was inhibited by 10% FBS in buffer wash solution (BWS). BWS contained 90 mM NaCl, 30 mM KCl, 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.02 mM EDTA, 32 mM sucrose, 0.015 mM neostigmine and 10% FBS, pH 7.4. For further preparation cells were washed twice with BWS (FBS free). Subsequently, cells for ACh measurement were resuspended in 80% acetonitrile containing 0.015 mM neostigmine and the cells for protein quantification taken from the same tube were resuspended in 10 mM HEPES (pH 7.4), 2 mM EDTA, 0.1 mM PMSF and 1 mM BZ. Acetylcholine content was measured by fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) as described previously .
All values are given as mean +/- standard deviation. Significant differences between the means were calculated by analysis of variance (ANOVA) followed by Scheffé-test.