Acc 034

The expression of TRPV4 channel protein in freshly dissociated FHH rat cerebral arterial myocytes was examined by Western blot analysis. Protein samples were prepared from freshly dissociated FHH rat cerebral arterial myocyte, and also from whole brains of FHH rat and the control Sprague Dawley rat that display normal myogenic cerebral autoregulation, by homogenization in lysis solution (Camiolo buffer, 75 mM potassium acetate, 300 mM NaCl, 10 mM EDTA, 100 mM l-arginine basic salt, and 0.25% Triton-X 100, protease inhibitor mix). Protein concentrations were determined using the Bradford assay [25 (link)]. Proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a membrane (Immobilon-P polyvinylidene difluoride, Millipore) at 25°C for 2 h at 15 V. Following 2 h blocking (with 5% skim milk in Tris-buffered saline with Tween-20 (TBST), the membrane was then incubated with polyclonal TRPV4 primary antibodies (ACC-034, 1:500, Alomone Laboratories) overnight at 4°C, and then with horseradish peroxidase (HRP)-conjugated goat anti-rabbit polyclonal secondary antibody (DakoCytomation; 1:1,500) for 1 h at room temperature. An ECL Plus Detection kit (Amersham Biosciences) was used to visualize bands.

Rabbit TRPV4 antibody (1:250; catalog number ACC034; Alomone Labs), rabbit phosphorylated-eNOS (p-eNOS) antibody (1:1,000; catalog number PA5-35879; Thermo Fisher Scientific), rabbit total eNOS antibody (1:100 to 500; catalog number NB300-500; Novus Biologicals), rabbit α-SMA antibody (1:100; catalog number ab5694; Abcam), GSK1016790A (catalog number G0798-10MG; Sigma Aldrich), GSK2193874 (catalog number 5106; Tocris), NONOate (catalog number 136587–13-8; Cayman Chemicals), and pan-NOS inhibitor l-NAME (N5751-5G; Sigma Aldrich) were used. Primary antibodies for TRPV4 and eNOS (total and phosphorylated) were validated using ocular tissues from TRPV4 and eNOS knockout mice (SI Appendix, Fig. S8). In addition, the same TRPV4 antibody was used in our previous study (54 (link)).

Immunohistochemistry staining was performed as previously described [19 (link)]. Primary antibodies against TRPV4 (ACC-034, alomone labs, Israel) and ZEB1(#70512, Cell Signaling Technology, MA, USA) were used. All staining was assessed by pathologists blinded to the origin of the samples and patient outcomes. The widely-accepted German semi-quantitative scoring system was used to assess the staining intensity and proportion of stained cells. Each specimen was assigned a score according to the intensity of staining (0, none; 1, weak; 2, moderate; 3, strong) and the proportion of stained cells (0, 0%; 1, 1–24%; 2, 25–49%; 3, 50–74%; 4, 75–100%). The final score for immunoreactivity was determined by multiplying the intensity by the proportion, ranging from 0 to 12.

Fixed colonic mucosal samples were dehydrated, paraffin embedded, and sectioned. Tissue sections of 4 μm thickness were mounted on silicone-coated slides, deparaffinized, and heated in citrate buffer (pH 6) using high-pressure cooking for antigen retrieval. Then, the sections were incubated with primary antibodies of TRPV1 (ab3487, 1 : 50, Abcam, Cambridge, MA, USA), TRPA1 (ACC-037, 1 : 50, Alomone Labs, Jerusalem, Israel), TRPV4 (ACC-034,1 : 50), TRPM2 (ACC-043, 1 : 50), and TRPM8(ACC-049,1 : 200) at 4°C overnight. After being rinsed with phosphate buffer saline (PBS) for three times, they were incubated with goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 30 minutes at room temperature and visualized with a diaminobenzidine-enhanced liquid substrate system (Sigma-Aldrich, St. Louis, MO, USA). For semiquantitation, captured images were analyzed with ImageJ software (National Institutes of Health).

Using immunblot methods as previously described [21 (link), 34 (link)], two times Laemmli Sample Buffer (Bio-Rad) with 5% fresh β-mercaptoethanol was mixed with 1 part of cell pellet, and then heated for 10 min at 95°C with votexing every 3 min during the heating process. The sample was centrifuged at 12,000 rpm for 2 min. The supernatant was laded to 4–15% SDS-PAGE gradient gel, transferred to PVDF membrane, and blocked with 3% nonfat milk for 1 hour at RT. Membranes were incubated with anti-TRPV4 (Alomone, ACC-034), anti-SK1 (Alomone, APC-039), anti-SK3 (Alomone APC-025), anti-IK1 (ThermoFisher PA5-33875) and anti-BKα (Alomone APC-021) antibody at 1:1000, respectively, at 4°C overnight. After washing, membranes were incubated with goat anti-rabbit secondary antibody (Santa Cruz Biotechnology) 1: 2000 for 1 h. All studies were performed in duplicate or triplicate.

mCCDcl1 cells were seeded on to Corning Costar^™ Transwell^™ Permeable Supports (0.4 μm pore size) in 24 well plates at 1×10⁵ cells/cm² in complete growth medium. After 7–9 days, cell monolayers were washed in PBS for two times and fixed in 100% methanol (chilled at -20°C) for 5 min at RT, followed by three times wash in ice cold PBS for 5 min each as done before [27 (link), 34 (link)]. The cells were then blocked in blocking buffer (PBS, 10% goat serum and 0.05% Triton X-100) for one hour. Whereupon primary antibodies were add to the blocking buffer at 1:100 dilution and incubated overnight at 4°C. The following primary antibodies were used: anti-SK1 (Alomone, APC-039), anti-SK3-ATTO-594 (Alomone, APC-025-AR), anti-IK1 (Alomone, ALM-051), anti-BKα (Alomone, APC-021) and anti-TRPV4 (Alomone, ACC-034). After primary antibody incubation, the cells were washed 3 times in PBS, 5 min each, followed by incubation with secondary antibody (Life Technologies, alexa fluor 488 or 594 labeled goat anti-rabbit) in blocking buffer. The cells were then washed another three times in PBS, 5 min each, and mounted with ProLong Gold Antifade Mountant with DAPI (Life Technologies). Cells were imaged at 100x using a Nikon A1R Confocal Laser Microscope or a Zeiss Axioskop 40 microscope equiped with a AxioCam MRm CCD camera.