(Kontes glass, Germany). Cell extract was further centrifuged at 16 K (25000 g) for 30 minute at 4uC and the clear supernatant was used for IP. Similarly, DRG tissues (L1-L6) from 2 male rats were isolated, homogenized in IP buffer. The lysate was centrifuged at 25.000 g, 30 min at 4uC and the clear extract was used for the further IP. Clear cell extract (800 ml equivalent to 1 mg of protein) and the corresponding antibody were applied to the equilibrated protein G beads. The mixture was incubated at 25uC for 4 hours on a shaker. After that the beads were washed 3 times, each time with 400 ml of IP buffer. A Hamilton syringe was used for all washing steps. Finally the beads were taken in 100 ml of IP buffer and 50 ml of 5x Laemmli sample buffer was added. The samples were boiled and used for western blot analysis. Expression and purification of MBP-fusion proteins Expression and purification of MBP-TRPV4-Ct (C-terminal cytoplasmic domain of TRPV4 fused with MBP) as well as of MBP-LacZ (LacZ fused with MBP) was based on a protocol described previously [36]. In short, the expression constructs were introduced into the Escherichia coli (E. coli) strain BL21DE3 by transformation heat shock method. Fusion protein expression was induced by addition of isopropyl thiogalactoside (IPTG) for 2 h. Cells were lysed by repeated freeze-thaw cycles in lysis buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20, lysozyme, benzonase and protease inhibitor cocktail). The lysed extracts were cleared by centrifugation (100000 g in a TFT 45 rotor for 2 h) and applied to amylose resin. The resins with bound proteins were washed thoroughly and finally the proteins were eluted with 10 mm maltose in elution buffer (50 mM PIPES, pH 6.8, 100 mM NaCl, 1 mM EGTA and 0.2 mM MgCl2). Pull down assays MBP-LacZ and MBP-TRPV4-Ct constructs were expressed in E. coli; the cleared cell lysates were applied to amylose resin (NEB), and incubated for 1 hour at RT (25uC) followed by washing. The amylose resin with bound proteins was resuspended in PEM-S buffer (50 mM PIPES, pH 6.8, 100 mM NaCl, 1 mM EGTA and 0.2 mM MgCl2). Approximately 50 ml of amylose resin per tube with the bound fusion protein was used for pull-down experiments. Depending on the respective experiment, the resin with coupled fusion protein was incubated with either 50 ml of soluble tubulin (1 mg/ml protein), 20 ml of soluble actin (0.1 mg/ml), 500 ml of soluble brain extract (1 mg/ml), or 500 ml of soluble F11 extract (1 mg/ml) for 1 hour at RT in the presence or absence of Ca2+ (2 mM). This was followed by three washes with 200 ml PEM-S buffer. The proteins were eluted by 10 mm maltose in 100 ml solution. Eluted samples were analysed by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). Co-sedimentation assay with polymerised actin, Taxol- stabilized microtubules (MT) and analysis for microtubule stability For experiments aimed at understanding the association of TRPV4-Ct with polymerised actin filaments, purified MBP- TRPV4-Ct alone was subjected to centrifugation at 70000 g/ 30 min/4uC to exclude any aggregates. The soluble MBP- TRPV4-Ct was further processed for all co-sedimentation assays. Either 5 mg of purified MBP-TRPV4-Ct or MBP alone in total 50 ml of PEM-S buffer or only 50 ml of PEM-S buffer was added to a saturated actin solution (50 mg actin in total 50 ml) in centrifuge tubes. ATP at a final concentration of 2.5 mM was added to each tube. The reaction tubes were incubated at 37uC for 30 minutes followed by centrifugal separation of pellet TRPV4 Regulates Cytoskeleton (polymerised actin filaments) and supernatant (free dimer) at 70000 g/30 min/37uC. Approximately 100 mg of purified ab-tubulin dimer in a total volume of 100 mL were incubated in modified PEM buffer (20 mm PIPES, pH 6.8, 0.2 mm MgCl2 and 1 mm EGTA supplemented by 1 mm Taxol and 5 mm GTP) for 30 min at 37uC, to form MTs. After that the MT were isolated by centrifugation at 70 000 g/30 min/37uC. Purified MBP-TRPV4- Ct or MBP only (each 5 mg) were incubated with Taxol-stabilized MT for 40 min at 37uC followed by centrifugal separation of pellet (MT) and supernatant (free dimer) at 70 000 g/30 min/37uC. In another experiment, MT was formed under Taxol-free conditions. Tubulin dimer (100 mg) with purified MBP-TRPV4-Ct or MBP only (5 mg each) were taken in total 100 ml PEM-S buffer with 5 mm GTP in the absence of Taxol and incubated for 30 min at 37uC. After that the MT and bound proteins were separated by centrifugal separation of pellet (MT) and supernatant (free dimer) at 70000 g/30 min/37uC. For experiments aimed at understanding the effect of TRPV4- Ct on MT stabilization, either 5 mg of purified MBP-TRPV4-Ct or MBP alone in total 50 ml of PEM-S buffer or only 50 ml of PEM-S buffer was added to a tubulin solution (5 mg tubulin in total 50 ml) in centrifuge tubes. Depending on the experimental conditions, MT-depolymerizing agent Nocodazole (final concen- tration 10 mM), Ca2+ (final concentration 0.1 mM) or both Nocodazole and Ca2+ was added to the tubes. GTP at a final concentration of 2.5 mM was added to each tube. The reaction tubes were incubated at 37uC for 30 minute s followed by centrifugal separation of pellet (MT) and supernatant (free dimer) at 70000 g/30 min/37uC. Purification of tubulin ab-tubulin dimers were purified from porcine brain as described previously [36]. In brief, from soluble brain extract tubulin was enriched by two cycles of polymerization in the presence of glycerol and GTP and depolymerization by cold temperature (ice cold), which were then followed by chromatography on phospho- cellulose. Immunocytochemistry For immunocytochemical analysis, cells were washed once with PBS and fixed with 2% PFA/PBS if not mentioned otherwise. Alternatively, where mentioned, an equal volume of 4% PFA was carefully added to the medium without disturbing the cell culture within the incubator to avoid morphological changes due to the PBS wash. The cells were fixed with PFA for 5 minutes at room temperature (25uC). For experiments aimed to explore the integrity of the cytoskeleton after TRPV4 activation, soluble components were removed by digitonin extraction as described before [31]. Briefly, after activation the cells were extracted with an isotonic cell membrane-permeabilization buffer (digitonin (50 mg/ml), PIPES (50 mM pH 6.8), EGTA (1 mM), MgCl2 (0.2 mM) and complete protease inhibitors (Roche) that perforates the cell membrane and allow all the soluble cellular proteins to diffuse out while insoluble cytoskeleton and cell morphology remain intact. After extraction the cells were quickly fixed with 2% PFA. Fixed cells were permeabilized with 0.1% Triton X-100 in PBS (5 min) and subsequently blocked with 5% normal goat serum or 5% BSA. For co-immunostaining of TRPV4 and tubulin, a rabbit polyclonal anti TRPV4 antibody (Sigma Aldrich) and a rat monoclonal anti-tyrosinated tubulin antibody (clone YL1/2) were used. For staining of actin filaments, either alexa-594 or alexa-488 labelled Phalloidin (molecular probe) was used. Alexa 594-labelled PLoS ONE | www.plosone.org 12 July 2010 | Volume 5 | Issue 7 | e11654