J Physiol 587.22 PIP3 and PIP2 activation of vascular TRPC1 5363 v.9.0 software with events lasting for <6.664 ms (2 × rise time for a 100 Hz, −3 db, low pass ﬁlter) being excluded from analysis. Single channel current amplitude histograms were plotted and ﬁtted with Gaussian curves with the peak of these curves determining the unitary amplitude of the single channel currents. Open probability (NPo) was calculated automatically using pCLAMP 9. Figure preparation was carried out using Origin 6.0 software (OriginLab Corp., Northampton, MA, USA) where inward single channel openings are shown as down- ward deﬂections. Solutions and drugs In cell-attached patch experiments the membrane potential was set to approximately 0 mV by perfusing cells in a KCl external solution containing (mM): KCl (126), CaCl2 (1.5), Hepes (10) and glucose (11), pH adjusted to 7.2 with 10 M KOH. Nicardipine (5 μM) was also included to prevent smooth muscle cell contraction by blocking Ca2+ entry through voltage-dependent Ca2+ channels. The bathing solution used in inside-out experiments (intracellular solution) contained (mM): CsCl (18), caesium aspartate (108), MgCl2 (1.2), Hepes (10), glucose (11), BAPTA (1), CaCl2 (0.48, free internal Ca2+ concentration approximately 100 nM as calculated using EQCAL software), Na2ATP (1) and NaGTP (0.2), pH 7.2 with Tris. The patch pipette solution used for both cell-attached and inside-out patch recording (extracellular solution) was K+ free and contained (mM): NaCl (126), CaCl2 (1.5), Hepes (10), glucose (11), TEA (10), 4-AP (5), iberiotoxin (0.0002), 4,4 -diisothiocyanatostilbene-2,2 -disulfonic acid (DIDS) (0.1), niﬂumic acid (0.1) and nicardipine (0.005), pH adjusted to 7.2 with NaOH. Under these conditions voltage-dependent Ca2+ currents, K+ currents, swell-activated Cl− currents and Ca2+-activated Cl− conductances are abolished and non-selective cation currents could be recorded in isolation. Anti-TRPC1 (which detects TRPC1 proteins with a predicted molecular mass of ∼100 kDa) and anti-PIP2 antibodies (which detect liposome complex of PIP2 molecules with a predicted molecular mass of ∼75 kDa, see manufacturer’s data sheet and Fukami et al. 1988) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and alomone labs (Israel), anti-PIP3 antibodies were from MBL (Japan) and anti-β-actin antibodies were from Sigma (UK). Pre-incubation of anti-TRPC1 anti- bodies with its antigenic peptide was carried out in a 1 : 2 ratio for at least 2 h in control experiments. Unless otherwise stated all other drugs were purchased from Calbiochem (UK), Sigma (UK) or Tocris (UK) and agents were dissolved in distilled H2O or DMSO (0.1%). DMSO alone had no effect on channel activity. The values are C 2009 The Authors. Journal compilation C 2009 The Physiological Society the mean of n cells ± S.E.M. Statistical analysis was carried out using paired (comparing effects of agents on the same cell) or unpaired (comparing effects of agents between cells) Students’ t test with the level of signiﬁcance set at P < 0.05. Immunoprecipitation and Western blotting Dissected tissues were ﬂash frozen and stored in 10 mM TRIS-HCl (pH 7.4) at −80◦C for subsequent use. Tissues were defrosted and mechanically disrupted with an Ultraturrax homogeniser and further disrupted by sonication on ice for at least 2 h. Tissues were subsequently centrifuged at 25 000 g for 30 min at 4◦C and the super- natant was discarded. The total cell lysate (TCL) was then collected by centrifugation at 11 200 g for 10 min in 10 mg ml−1 RIPA lysis buffer (Santa Cruz Biotechnology), supplemented with protease inhibitors. Protein content was quantiﬁed using the Bio-Rad protein dye reagent (Bradford method). TCL was retained on ice for subsequent experimental procedures including dot-blots and immunoprecipitation. Dot-blots were carried out by ‘spotting’ 2–5 μl of TCL on prepared immobilon-p poly- vinylidene diﬂuoride (PVDF) membranes. Membranes were allowed to dry prior to detection using conventional Western blotting techniques (see later). The immuno- precipitation protocol was carried out using the Millipore Catch and Release R kit, where spin columns were loaded with 500 μg of TCL and 2–6 μg of antibody and immuno- precipitated for 2 h at room temperature. Immunoprecipitated samples were eluted with Laemmli sample buffer and incubated at 60◦C for 5 min. One-dimensional protein gel electrophoresis was performed in 4–12% Bis-Tris Gels in a Novex mini-gel system (Invitrogen) with 10–20 μg of total protein loaded in each lane. Separated proteins were trans- ferred onto PVDF membranes using the Invitrogen iBlot apparatus. Western blotting was subsequently carried out on membranes which were incubated with the appropriate primary antibody for 2 h at room temperature. Where possible, alternative antibodies raised against different epitopes were used for immunoprecipitation and Western blot analysis. Following antibody removal membranes were washed for 2 h with milk/phosphate-buffered saline with Tween 20 (PBST) and were subsequently incubated with horseradish peroxidase-conjugated secondary anti- body diluted 1 : 1000–5000 in milk/PBST. Membranes were then washed 3 times for 15 min in PBST, followed by a ﬁnal wash in PBS before being treated with ECL chemiluminescence reagents (Pierce Biotechnology, Inc., Rockford, IL, USA) for 1 min and exposed to photographic ﬁlms. Data shown represents n values of at least three separate experiments.