Trpv4

Cells were seeded in 12-well plates and cultured in a medium supplemented with 10% FBS for 24 h. After fixing with 4% paraformaldehyde for 30 min, the cells were blocked with 1.5% bovine serum albumin (BSA) for 1 h. Then, cells were incubated with primary antibodies against TRPV4 (1:2000, Abcam, Cambridge, MA, USA), phospo-cortactin (1:1000, Affinity, China), and cofilin (1:1000, Affinity, China) at 4 °C overnight followed by incubation with FITC-labeled goat anti-rabbit IgG for 1 h at room temperature. DAPI (Beyotime, China) was used to stain the cell nucleus. All the images were captured with the fluorescence microscope (Zeiss, Axio vert A1, Germany).

The temporal bones were removed and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 1.5 hr at 4°C. The temporal bones were decalcified by incubation in 10% EDTA at 4°C for 3–5 days. The EDTA solution was changed daily. The bones were then embedded in the OCT compound for cryostat sectioning. The sections of 10 μm thickness were washed in PBS, and nonspecific binding was blocked with 1% bovine serum albumin (BSA) and 10% goat serum in PBS plus 0.1% Triton X-100 (PBST) for 1 hr. The primary antibodies, chicken anti-Tuj1 (Abcam), mouse anti-myelin basic protein (Abcam), rabbit anti-Myo7a (Proteus Biosciences, Inc), mouse anti-Tuj1 (Abcam), rabbit anti-TRPA1 (Abcam), TRPC3 (Novus Biologicals), TRPC6 (Abcam), TRPV1 (Novus Biologicals), TRPV4 (Abcam), were incubated overnight at 4°C. After incubating the primary antibodies, the slides were washed three times with PBST and incubated with secondary antibodies for 1.5 hr at RT in the dark. We used Alexa Fluor 647-conjugated goat anti-mouse and Cy3-conjugated goat anti-chicken, Alexa Fluor 488-conjugated goat anti-rabbit, Alexa Fluor 568-conjugated goat anti-mouse (Jackson ImmunoResearch Labs) in a dilution of 1:500. Other markers used were phalloidin-Fluor 647 (Abcam) for F-actin and DAPI (Sigma) for nuclear stain. The slides were then examined under a confocal microscope (LSM 510, Zeiss).

TRPV4, ROCK1, ROCK2, and paxillin (PXN) antibodies were purchased from Abcam (Cambridge, MA, USA). RhoA, RhoB, RhoC, CDC42, RAC1/2, LIMK, phospho-LIMK, cofilin, phospho-cofilin, MLC, phospho-MLC, MYPT, and phospho-MYPT antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). GAPDH were purchased from Proteintech (Rosemont, IL, USA). Rho pathway antagonist Y27632, calcium chelator BAPTA-AM, TRPV4 agonist GSK1016790A, and its antagonist HC067047 were purchased from Selleck (Shanghai, China). Opti-MEM medium and Lipofectamine RNAiMAX reagent, Fluo-4 AM, Rhodamine Phalloidin, puromycin, and DAPI were purchased from Invitrogen (Massachusetts, USA).