Phosstop and complete mini

Cdc20 complexes were purified from cells 8 h after release from a 20-h thymidine block and harvested by trypsin treatment after thorough washing with PBS. About 24 h (cells expressing YFP, YFP–Cdc20^WT and YFP–Cdc20^Ala) or 8 h (cells expressing YFP–Cdc20^Asp) prior to harvest, the expression of the indicated constructs was induced using 1–2 ng ml⁻¹ doxycycline. Cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.8, 1 mM DTT, 0.1–1% NP40 and 0.5 U μl⁻¹ Benzonase) supplemented with PhosStop and Complete Mini (Roche) with a volume correlated to the cell pellets weight. Cdc20 complexes were immunoprecipitated without clearance of the lysate using a mouse monoclonal Cdc20 antibody cross-linked to Protein G-Sepharose 4B (Life Technologies) or YFP affinity resin (Chromotek) overnight at 4 °C. Precipitated protein complexes were washed in lysis buffer after precipitation of beads at 30g for 30 s and eluted in 4 × SDS sample buffer (Life Technologies). For Calyculin A (Cell Signalling) treatment, the drug was added for 30 min prior to harvest after 8 h at a concentration of 100 nM.

Cells were washed with ice cold PBS and lysed in a buffer containing 50 mM Tris-HCl (pH 7.6), 137 mM NaCl, 0.5 mM DTT, 1% Nonidet-P40, 0.2% sodium dodecyl sulphate (SDS), 0.5 μM Okadaic acid, and Phosphatase and Protease Inhibitor Cocktail Tablets (PhosSTOP and cOmplete Mini, Roche). Protein samples (100 μg for microglial lysates and 50 μg for astroglial lysates) were dissolved into 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and wet-transferred overnight at 4°C to a nitrocellulose membrane (Whatman, GmbH, Dassel, Germany). Membranes were blocked with 5% (w/v) dry skimmed milk or BSA in TBS with 0.1% Tween 20 (TTBS) for 1 h at room temperature and incubated overnight at 4°C with the corresponding primary antibody (for more information, see Table 
3). After washing with TTBS and TBS, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature and the protein bands were detected using Supersignal west pico or west femto chemiluminescent substrate (Pierce, Rockford, IL, USA).
Image densitometry was performed with a Bio-Rad GS-810 scanner (BIO-RAD Labs, Richmond, CA, USA) and analyzed with Quantity One 4.2 software (BIO-RAD). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and α-actinin expression were used as loading control for microglia and astrocyte samples, respectively.

Jurkat (clone E6-1; American Type Culture Collection, Manassas, VA, USA) cells and GM12155 (Coriell Institute, Camden, NJ, USA) cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2.05 mmol/L L-glutamine, 100 units/mL penicillin, 100 mg/mL streptomycin, and 1 mmol/L sodium pyruvate at 37 °C with 5% CO₂. HEK293T (American Type Culture Collection, Manassas, VA, USA) cells were cultured in DMEM medium supplemented with 10% FBS, 100 units/mL penicillin, 100 mg/mL streptomycin, and 1 mmol/L sodium pyruvate at 37 °C with 5% CO₂.
Whole-cell lysates were extracted using EBC lysis buffer (50 mM Tris-HCl, pH 7.5, 120 mM NaCl, 0.5% Nonidet P-40 [NP-40], and 1 mM EDTA) containing protease and phosphatase inhibitors (cOmplete Mini and PhosSTOP; Roche Diagnostics GmbH, Mannheim, Germany) as previously described [47 (link)].

For cultured cells, RNA was extracted using the NucleoSpin RNA Plus kit (Macherey-Nagel #740984). In some experiments, DNA, RNA and proteins were extracted using the NucleoSpin TriPrep kit (Macherey-Nagel #740966), following the manufacturer’s instructions. For RNA extraction from tissues, samples were first homogenized in LPB buffer from the NucleoSpin RNA Plus kit using a Buller Blender Gold apparatus (Next Advance) and 0.5 mm zirconium oxide beads. For protein extraction from tissues, samples were sonicated in RIPA buffer (10 mM Tris-HCL pH8.0, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, supplemented with protease and phosphatase inhibitors (Complete Mini and PhosSTOP, Roche)) in 1.5 mL microtubes on ice with five 5-sec pulses at power3 on a Misonix XL-2000 instrument, with a 10-sec timeout on ice between each pulse. The lysates were further incubated with rotation for 30 minutes at 4°C, spun at 11000 x g at 4°C for 15 minutes, and the supernatants transferred to new 1.5 mL microtubes. RNA and protein samples were stored at -80°C until further analysis.

Stably transfected HEK 293 cells were seeded onto poly-l-lysine-coated 60 mm dishes and grown to 80% confluence. The cells as well as freshly dissected tissues from WT (C57BL6) and VPAC2-deficient mice, including small and large intestine, gastric mucosa, brain, pancreas, spleen and testis, were lysed in detergent buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 10 mM NaF, 10 mM disodium pyrophosphate, 1% Nonidet P-40, 0.5% sodium deoxycholate and 0.1% SDS) in the presence of protease and phosphatase inhibitors Complete Mini and PhosSTOP (Roche Diagnostics). The receptors were enriched using wheat germ lectin agarose beads as described previously (29, 30) (link). Proteins were eluted from beads using SDS sample buffer for 20 min at 45 °C. The samples were then subjected to 7.5% SDS–polyacrylamide gels, and blotted onto PVDF membranes. The membranes were incubated with the rabbit monoclonal anti-human VPAC1 antibody (SP234; dilution 1:500) or the rabbit monoclonal anti-human VPAC2 antibody (SP235; dilution 1:500), followed by incubation with peroxidase-conjugated secondary anti-rabbit antibody (Santa Cruz Biotechnology; dilution 1:5000) and ECL detection (Amersham). For adsorption controls, antibodies were preincubated with 10 μg/ml of their cognate peptides for 2 h at room temperature.