Multi gauge ver 3.2 imaging software

Whole cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer containing 1× protease and phosphatase inhibitor cocktail (Thermoscientific, CA, USA) RIPA buffer was composed of 150 mM Sodium chloride, 1% Triton X-100, 0.5% Sodium deoxycholate, 0.1% SDS and 50 mM Tris adjusted to pH 8.0. After lysing cells with RIPA buffer, they were centrifuged at 16200 g for 15 min and supernatants containing soluble proteins were collected. Protein contents were measured using BCA protein assay reagent (Pierce, Rockford, IL, USA). Equal amounts of total proteins were then separated by SDS-PAGE and transferred onto Hybond ECL nitrocellulose membranes (Amersham Life Science, Buckinghamshire, UK) at 200 mA for an hour. Membranes were blocked in 5% skim milk in Tris-buffered saline (TBS)-Tween 20 (TBS-T) at room temperature for 1 h, incubated in skim milk-TBS overnight at 4 °C, washed three times with TBS-T, and incubated with horseradish peroxidase-conjugated secondary antibody in skim milk-TBS for 1 h at room temperature. Immunoreactive proteins were visualized using an ECL kit (Pierce, Rockford, IL, USA) and digitally processed using LAS-4000 mini (Fuji, Japan). Membranes were stripped and reprobed with an actin antibody as a loading control. Densitometric analysis of blots was performed with Multi Gauge Ver 3.2 imaging software in a Fuji Image Station.

HUVECs were pretreated with indicated compounds for 1 h prior to treatment with 5-HT and whole lysates were prepared using RIPA buffer containing proteases and phosphatase inhibitors. BCA protein assay reagent (Pierce, Rockford, IL, USA) was used for quantitation of the protein content. Equal amounts of protein were separated by SDS-PAGE and transferred at 200 mA onto Hybond ECL nitrocellulose membranes (Amersham Life Science, Buckinghamshire, UK) for 1 h. The membranes were blocked in 5% bovine serum albumin (BSA) and 5% skim milk in Tris-buffered saline (TBS)-Tween 20 (TBS-T) for phosphorylated and total protein, respectively, at room temperature for 1 h followed by incubation with specific antibodies in BSA or skim milk-TBS at 4°C overnight. The membrane was then washed three times with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody in BSA or skim milk-TBS for 1 h at room temperature. Immunoreactive proteins were detected using an ECL kit (Pierce) under a luminescent image analyzer, LAS-4000 mini (Fuji, Japan). The membranes were reprobed with β-actin antibody for loading control. The protein density was analyzed using Multi Gauge Ver 3.2 imaging software in a Fuji Image Station.