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For immunocytochemistry, cells were plated on a glass‐bottom chamber slide (Matsunami Glass, SCS‐008, Osaka, Japan). After fixation, cells were washed, and permeabilized with 0.5% Triton X‐100. Subsequently, cells were washed, and incubated with blocking buffer that contained 5% goat serum and 1% BSA. After blocking, cells were incubated with the primary antibodies, mouse anti‐vimentin antibody (Sigma, V6389, St. Louis, MO, 1:20 dilution), mouse anti‐E‐cadherin antibody (BD Biosciences, 610182, San Jose, CA, 1:50 dilution), rabbit anti‐phospho‐paxillin antibody (Cell Signaling Technology, 2541, Danvers, MA, 1:20 dilution) and mouse anti‐vinculin antibody (Sigma, V9131, 1:200 dilution). Cells were washed, and stained with the secondary antibodies, rabbit anti‐mouse IgG antibody conjugated to Alexa 568 (Life Technologies, A11061, Carlsbad, CA, 1:200 dilution), goat anti‐rabbit IgG antibody conjugated to Alexa Fluor 546 (Life Technologies, A11010, 1:1000 dilution) and goat anti‐mouse IgG conjugated to Alexa Fluor 488 (Life Technologies, A11001, 1:1000 dilution). In cells immunostained for vimentin and E‐cadherin, nucleus was counterstained with DAPI. Fluorescent images were obtained using an all‐in‐one microscope (Keyence, BZ‐9000, Osaka, Japan).

For transfection, HEK293 cells were plated on poly-lysine coated chamber slides (SCS-008, Matsunami, Japan) in DMEM medium containing 10% FBS. HEK293 cells on chamber slide at 80% confluency were transfected with plasmids expressing N-terminal FLAG-tagged mPRβ, C-terminal His-tagged mPRβ, or N-terminal FLAG-tagged GPR41. Briefly, 1 μg of plasmids were added in 50 μL Opti-MEM I medium. Lipofectamine 2000 (2 μL) (Invitrogen) were separately prepared in 50 μL Opti-MEM I medium and incubated for 5 min at room temperature. The two solutions were mixed, and then incubated for 20 min at room temperature. This mixture was added to HEK293 cells and the cells were incubated overnight at 37 °C in a 5% CO₂ incubator.
The cells were fixed in 4% formaldehyde in PBS for 10 min at room temperature and incubated with 0.1% Triton-X in PBS or PBS alone for 5 min at room temperature. After washing with PBS, the cells were pre-incubated for 1 h in 1% BSA in PBS, and then probed with the Alexa488-conjugated mouse anti-His-tag antibody (MBL, Japan) at a dilution of 1:200 in 1% BSA in PBS or Alexa488-conjugated mouse anti-FLAG antibody (MBL) at a dilution of 1:200 in 1% BSA in PBS for 1 h at room temperature. After washing twice with PBS, the cells were observed using a Zeiss LSM700 confocal microscope.

Cells cultured on chamber slides (Matsunami Glass, SCS-008) were fixed in 4% paraformaldehyde for 8 min at room temperature. The fixed cells were permeabilized with 0.1% Triton X-PBS for 15 min at room temperature and blocked with 3% BSA in 0.1% Tween-20 TBS (3% BSA-TTBS) for 15 min at room temperature. Next, the cells were incubated with primary antibodies diluted in 3% BSA-TTBS for 1 hr at room temperature, washed twice with PBS, and incubated with secondary antibodies conjugated with Alexa568, 647 Fluor (Invitrogen) diluted in 3% BSA-TTBS for 1 hr at room temperature. The nuclei were counterstained with DAPI and observed under a confocal microscope (Olympus, FV1000) at the Support Unit for Bio-Material Analysis in the RIKEN BSI Research Center (RRC).