Phosphatase inhibitor cocktail edta free

The rats were deeply anesthetized with pentobarbital (60 mg/kg, i.p.) on days 0, 7, or 14 after the start of PTX (4 mg/kg) treatment, and spinal cords were removed for Western blot analysis. Spinal cords were homogenized in cold 1× RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA) containing phosphatase inhibitor cocktail (EDTA free) (Nacalai Tesque, Inc. Tokyo. Japan) and protease inhibitor cocktail (Nacalai Tesque). The homogenates were centrifuged at 4 °C for 30 min at 15,000× g, and the supernatants were collected. Spinal cord protein (30 μg) was separated by an SDS-polyacrylamide gel (7.5%) and transferred onto polyvinylidene difluoride membranes. Anti-TRPV1 antibody (1:200, Alomone Labs, Jerusalem, Israel) was used for Western blotting, with anti-β-actin (Sigma-Aldrich, 1:1000) used as the internal control. Horseradish peroxidase-labeled goat anti-rabbit immunoglobulin G (IgG) (Santa Cruz Biotechnology, 1:5000) was used as the secondary antibody. Specific bands were detected by enhanced chemiluminescence (ECL) with the ECL^TM Prime Western Blotting Detection kit (GE Healthcare, Wauwatosa, WI, USA) using a luminescent image analyzer LAS4000 (Fuji Film, Japan). The intensities of immunoreactive bands were quantified using MultiGage Ver.3 software (Fuji Film, Tokyo, Japan).

Fucoxanthinol was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Cycloheximide and Phosphatase Inhibitor Cocktail (EDTA free) were purchased from Nacalai Tesque (Kyoto, Japan). MG-132 was purchased from Peptide Institute (Osaka, Japan).

hAECs were lysed with PROPREP (iNtRON Biotechnology, MA, USA) supplemented with Phosphatase Inhibitor Cocktail (EDTA free) (Nakarai tesque, Japan). Protein extracts were analyzed by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS–PAGE; Bio-Rad Laboratories, CA, USA) followed by a Western blot assay. EzBlockChemi (ATTO, Japan) was used for blocking, and Western BLoT Immuno Booster (Takara, Japan) was used for the reaction. The primary antibodies were as follows: Smad1, Smad2/3, phospho-Smad1 (Ser463/465)/5 (Ser463/465)/9 (Ser465/467), and phospho-Smad2 (Ser465/467)/3 (Ser423/425) (Cell Signaling Technology, MA, USA) at a 1:1,000 concentration, and GAPDH (GeneTex, CA, USA) at a 1:10,000 concentration. The secondary antibodies were peroxidase AffiniPure goat anti-mouse IgG and anti-rabbit IgG (Jackson ImmunoResearch Laboratory, PA, USA) at a 1:20,000 concentration. Western blot images were developed with Western BLoT Hyper HRP Substrate (Takara, Japan) and taken with a LumiCube System (Liponics, Japan).