Ripa lysis buffer

For the in vitro study, LNCaP cells were treated with EGCG (20 µM) + Q (2.5 µM), Arc (1 µM), EGCG+Q+Arc, or the control; WPE1-NA22 cells were treated with EGCG (20 µM) + Q (2.5 µM), Arc (0.5 µM), EGCG+Q+Arc, or the control, for 24 h. The cells were harvested and total protein was extracted using an RIPA lysis buffer (Santa Cruz, CA, USA). For the animal study, total protein was extracted from the prostate/tumor tissues using the RIPA lysis buffer (Santa Cruz). The procedure for the Western blot analysis was described previously [38 (link)]. Briefly, 50 μg of protein was separated on a 4–12% Bis-Tris gel (Invitrogen), electrotransferred to nitrocellulose membranes, and blocked in 5% nonfat milk. The membranes were incubated with primary antibodies for the detection of AR (sc-7305, 1:500 dilution), PTEN (sc-7974, 1:500 dilution) (Santa Cruz Technology, Santa Cruz, CA, USA), Akt (4685, 1:1000 dilution), and p-Akt (4058, 1:1000 dilution) (Cell Signaling Technology, Danvers, MA, USA), respectively. GAPDH was used as a loading control.

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).

Placental and hippocampal tissues from sham and RUPP mice were homogenized using ChemCruz RIPA lysis buffer in a mini bead homogenizer at 4000 rpm. The supernatant for each sample was collected. A bicinchoninic acid (BCA) protein assay was used to determine the amount of protein in each sample. Samples were prepared for Western blot analysis using 1x PBS, 4X sample buffer which contained 2 beta-mercaptoethanol. Samples were electrophoresed through Criterion TGX 4–20% Stain-free gel at 120 V for 90 min. When completed, the gel was activated using the ChemiDoc MP Imaging System for 45 s. The proteins were transferred to nitrocellulose membranes using the Trans blot turbo transfer kit. Following 30 min of blocking in Odyssey blocking buffer, the primary antibodies for the proteins of interest (ASIC2a antibody, 1:1000, Thermo Fisher Scientific, catalog# OSR00097W) and alpha-tubulin antibody (1:15,000, Abcam, catalog# ab89984) were added and incubated overnight. After washing, the secondary antibodies, donkey anti rabbit (1:15,000, Li-Cor, Catalog# 131591), and donkey anti chicken (1:15,000, Li-Cor, Catalog# 925-68028) were added. Membranes were imaged using the Chemidoc MP system. Protein expression was normalized to alpha tubulin expression. Blots were analyzed using the Bio-Rad Chemidoc Image Lab software.

Cell protein extraction and western blot were conducted as described previously [30 (link), 35 (link)]. Protein was extracted from cells using 1x RIPA lysis buffer (Santa Cruz, Dallas, TX). Samples were electrophoresed and subjected to western blot using primary antibodies against RGS2 (Proteintech, Chicago, IL) and β-actin. IRDye-800 secondary antibodies (LI-COR, Lincoln, NE) were used to capture images with a LI-COR Odyssey.