Irdye 800cw donkey anti rabbit secondary antibody

Western blot. HMVEC were collected and treated as follows: 1) For total protein extracts, cells were mechanically lysed with RIPA buffer (Santa Cruz) supplemented with protein inhibitors, and spun down at 10,000g for 10 min at 4 °C. 2) For membrane protein fractions, cells were sonicated in PBS pH 7.4 (with protease inhibitors) and spun down at 8,000g. Supernatant was spun down at 160,000g for 1 h at 4°C. Membrane pellets were solubilized with 2% SDS and spun down at 160,000g for 1 h at RT. Supernatants from total protein extract and membrane protein fractions were loaded in Mini-PROTEAN TGX Stain-free Precast gels and were exposed to UV for 45s before transfer. Anti-TRPV4 (1:200, Santa Cruz) and IRDye® 800CW Donkey Anti-Rabbit secondary antibodies (1:10,000, LI-COR) were used for western blots. Membranes were developed in a LI-COR and ChemiDoc Touch imaging system (Bio-Rad), for infrared and UV signals, respectively. Immunostaining. HMVEC were fixed with 4% paraformaldehyde for 15 min; permeabilization was achieved with 0.1% Triton X-100 in PBS for 15 min. HMVEC were incubated with primary anti-TRPV4 antibody (1:250; Alomone Cat # ACC-124) at 4 °C overnight. Samples were washed and incubated with goat anti-rabbit IgG secondary antibody conjugated to goat anti-rabbit IgG-TR (1:200 Cat # sc-2780). Fluorescence was detected with a Zeiss Axiophot 200 LSM Pascal.

Protein samples were boiled in 2x SDS sample buffer for 5 minutes and loaded into 4–20% Mini-PROTEAN TGX Precast Gels (Bio-Rad 456). If used for Western Blots, SDS gels where transferred to immobilon-P PVDF membranes (Milipore). Immunoblots were blocked for 1 hour with 1% non-fat dry milk in 1x TBS buffer containing 0.1% Tween-20 (TBST). The membranes were then probed overnight at 4°C with rabbit anti-FLAG antibody (Thermo Fisher Scientific 740001; 1:8000) diluted in 1% non-fat dry milk. Membranes were then washed three times with TBST and incubated with IRDye 800CW Donkey anti-Rabbit secondary antibodies (Li-cor 925–32213) for 1 hour at room temperature. The signal was visualized by Licor Odyssey CLx. If SDS gel was submitted for in-gel digestion and mass spectrometry (below), then gel was incubated in Coomassie blue stain overnight at room temperature and destained with DI water.