Pvdf membrane

The concentrations of proteins were determined by BCA assay (Beyotime, Nantong, China). Then proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a polyvinylidene fluoride (PVDF) membrane (Amersham, UK). Following blockage with 5% nonfat dry milk in PBS for 1 h, the blotting membranes were probed overnight, respectively, at 4 °C, with the TRPV4 primary antibodies, then they were probed with the appropriate HRP-conjugated secondary antibodies. The PVDF membrane was exposed to X-ray film and immunoreactive bands were detected by reaction with enhanced chemiluminescence (ECL) detection system reagents (Amersham, Arlington Heights, IL, USA). For loading control, the membrane was probed with a monoclonal antibody for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Lab Works Image Acquisition and Analysis Software (UVP, Upland, CA, USA) was used to quantify band intensities. Antibodies were purchased from Abcam (Cambridge, UK).

The ipsilateral sciatic nerve, L4-L5 DRGs, and corresponding dorsal horn of the spinal cord from the rats of the sham control and experimental groups were harvested as previously described [14 (link),15 (link),26 (link)]. Equal amounts (50 µg) of protein extracts were loaded and separated via SDS-PAGE using 8–12% acrylamide gradients. After electrophoresis, the separated proteins were transferred electrophoretically to a polyvinylidene difluoride (PVDF) membrane (Amersham Biosciences, Amersham, UK). Nonspecific sites were blocked via incubation of the membrane in blocking buffer (5% nonfat dry milk in T-TBS (TBS containing 0.05% Tween 20)) overnight. The membranes were incubated with the indicated primary antibodies (refer to Supplementary Table S2) for 1 h at room temperature. After electrophoresis, the separated proteins were transferred electrophoretically to a PVDF membrane (Amersham Biosciences, Amersham, UK). The raw materials, i.e., original uncropped and unprocessed scans of the blots in the sections of original images of blots and gels, were provided as per Supplementary Figure S3.
Nonspecific sites were blocked via incubation of the membrane in blocking buffer (5% nonfat dry milk in T-TBS (TBS containing 0.05% Tween 20)) overnight.

Proteins that were extracted from ox-LDL-treated cells were separated with Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (cat. no. P1200; SDS-PAGE, Solarbio) and polyvinylidene difluoride (PVDF) membrane (Amersham, Munich, Germany), and then PVDF membrane was blocked with TBST buffer containing 5% bovine serum albumin. The protein bands were incubated with primary antibodies: CyclinD1 (ab16663, 1:1000, Abcam, Cambridge, MA, USA), cleaved-caspase 3 (ab2302, 1:1000, Abcam), ROCK2 (ab125025, 1:1000, Abcam), GAPDH (ab9485, 1:1000, Abcam), and the secondary antibody (1:5000, ab205718, Abcam), respectively. Finally, the protein bands were incubated with ECL Western Blotting Substrate (cat. no. PE0010; Solarbio) and observed by a gel imaging analysis system. The original western blots were showed in Additional file 1.