Blocking one p solution

Proteins (100 μg/lane) were electrophoresed at 100 V for 2 hours using a 12⋅5 % SDS-PAGE gel for separation. Next, the proteins were transferred to a polyvinylidene difluoride membrane (PVDF) (Amersham Life Science Inc., Commonwealth of Massachusetts, USA) at 100 mA for 2 hours. The membrane was incubated with Blocking One or Blocking One-P solution (Nacalai Tesque, Kyoto, Japan) for 30 min, and then the primary antibody diluted 1:1000-500 with Can Get Signal Solution 1 (Toyobo, Osaka, Japan) was subjected to a reaction at 4°C overnight. The next day, the membrane was washed with TBST containing 1 M Tris-HCl (pH 7⋅5), NaCl and 20 % Tween 20, and then the anti-rabbit or mouse horseradish peroxidase-conjugated IgG diluted 1:10 000-2000 with Can Get Signal Solution 2 (Toyobo, Osaka, Japan) was subjected to an antibody reaction at room temperature for 1 hour.
Protein band detection was performed using Chemi-Lumi One Super (Nacalai Tesque, Kyoto, Japan) and Ez-Capture ST (ATTO Corporation, Tokyo, Japan). β-actin was used as a loading control. Protein band intensity analysis was conducted using ImageJ public domain software (National Institutes of Health, State of Maryland, USA)^{(26 (link),41 (link))}.

Differentiated type II and type III cells were identified using antibodies against gustducin, T1R3, PLCβ2 (type II cell markers) and CaIV (type III cell marker). Sections of the CVP were prepared in a similar manner to that for the cell proliferation assay. The sections were washed with TNT buffer three times for 5 min and preincubated with Blocking One-P solution (Nakalai Tesque, Kyoto, Japan) for 1 h at room temperature. Next, the sections were incubated with primary antibody against T1R3 (1:200; goat anti-T1R3, Santa Cruz Biotechnology, Dallas, TX, USA), gustducin (1:200; rabbit anti-Gα_gust(I-20), Santa Cruz Biotechnology, Dallas, TX, USA), PLCβ2 (1:200; rabbit anti-PLCβ2, Santa Cruz Biotechnology, Dallas, TX, USA) or CaIV (1:100; goat anti-CA4, R&D Systems, Minneapolis, MN, USA) in Blocking One-P solution overnight at 4 °C. The sections were washed with TNT buffer three times for 5 min and then incubated for 2 h at room temperature with appropriate secondary antibody in 1% blocking reagent: Alexa Fluor 488 donkey anti-goat IgG (Invitrogen, Carlsbad, CA, USA) for T1R3, Alexa Fluor 594 donkey anti-rabbit IgG (Invitrogen, Carlsbad, CA, USA) for gustducin and PLCβ2, and Alexa Fluor 568 donkey anti-goat IgG (Invitrogen) for CaIV.

The cells were washed in phosphate-buffered saline and lysed in a RIPA Lysis Buffer (EMD Millipore, Burlington, MA) containing complete Protease Inhibitor Cocktail and PhosSTOP (Roche Applied Science). The membranes were blocked in Blocking One or Blocking One-P solution (Nacalai Tesque, Kyoto, Japan). Western blotting was performed with standard methods. The ChemiDoc Imaging System with Image Lab 6.0 software (Bio-Rad, Hercules, CA) was used for visualisation.

We washed the cultured cells with PBS and extracted the total protein using a complete Lysis-M reagent (Roche, Basel, Switzerland) containing 1% Halt™ Phosphatase Inhibitor Cocktail (Thermo Fisher Science, Waltham, MA, USA) for 15 min at room temperature. We subsequently centrifuged the cell lysate at 15,000×g for 15 min at 4 °C, separated 7.5 µg of total protein in 10% or 4–15% gradient SDS-PAGE (Bio-Rad, Hercules, CA, USA), and transferred it to a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Burlington, MA, USA). Nonspecific binding to the PVDF membrane was blocked in Blocking one-P solution (Nacalai Tesque) at room temperature for 30 min. The first antibody was reacted at 4 °C for overnight. Subsequently, we washed the PVDF membrane in PBS-T and enhanced the protein signal by anti-mouse or rabbit IgG antibody (1/10,000 or 5000) at room temperature for 1 h. We used ECL select (Roche) or ECL Prime reagent (Roche) to detect protein signals. A densitometric analysis of western blot was performed using a densitometer (CS analyzer version 3.0 software, ATTO, Tokyo, Japan), and the intensity was normalized to the b-actin protein levels.