Albumin bovine 5

Cells were seeded on glass slide in 6-well dishes. The next day, these cells were washed three times with PBS. Thereafter, the cells were fixed and permeabilized by sequential incubation in 4% p-formaldehyde for 20 min at room temperature and in 0.1% Triton X-100 for 10min, followed by 30 min incubation in a blocking buffer (3% BSA). The actin cytoskeleton was visualized with a Rhodamine-phalloidin (5U/ml, Invitrogen) which need to incubate for 30 min. The nuclear were stained with DAPI for 20 min. For the detection of proteins, after being blocked by Albumin Bovine V (Biosharp), samples were incubated with primary antibody to FAK (1:200) at 4℃ overnight following secondary-antibody FITC (Beyotime). The nuclear were stained with DAPI for 20min. Photomicrographs were obtained using a fluorescence microscope.

BRL-3A cells treated with 1.2 mM NEFAs and siOrai1 were cultured on the bottom well (Cellvis, Hangzhou, China) for 24 h and then fixed with 4% paraformaldehyde for 30 min at room temperature. BRL-3A cells were incubated with 3% Albumin Bovine V (Biosharp, Hefei, China), 5% normal goat serum (Boster, Wuhan, China), and 0.5% Triton in PBS (Biofroxx, Guangzhou, China) for 30 min at room temperature for blocking unspecific bindings. Then, rabbit anti-Orai1 and mice anti-NFκB p65 were incubated in cells overnight at 4°C in a humidified chamber. The cells were incubated with Cy3 goat anti-rabbit IgG (1:500, Beyotime) and goat anti-mice IgG-FITC (Santa Cruz Biotechnology) for 1 h at room temperature, respectively, after rinsing four times with PBS. The nuclei were stained with Hoechst 33342 dye (Beyotime) for 30 min at room temperature. Fluorescence and images were obtained with a confocal laser-scanning microscope (Leica TCS SP8).

The materials are mini osmotic pump (American Health Medical Instruments International), blood pressure meter (OMRON, Japan), IonOPTIX (American IonOPTI company), bechtop (Suzhou Su Clean), inverted microscope (O-lympus, Japan), HVE-50 autoclave (Hiraya-ma, Japan), PA (Ca#H8780-100g, Solarbio Biological Co., Ltd.), bovine serum albumin (Albumin Bovine V, BSA, Biosharp-100G), and specific nNOS inhibitor (S-methy-L-thiocitrulline, SMTC) (100 μM). L-VNIO (vinyl-L-N-5-(1-imino-3-butenyl)-L-omithine) is a NOS1 inhibitor.