Rspo2

Human RSPO2 cDNA (clone ID 100062073) was purchased from Open Biosystems. To generate CMV-RSPO2, the coding region of RSPO2 cDNA was amplified from the clone by PCR (see Supplementary Table S1) and was inserted into the HindIII and Xbal sites of the CMV-driven expression vector, p3XFLAG-CMV-14 (Sigma-Aldrich). A 3198-bp promoter region (positions −3349 to +7 of the ATG translational start site) of the mouse Ckm gene encoding MCK and a 6398-bp promoter region (positions −6358 to +41 of the ATG translational start site) of the mouse Slc18a3 gene encoding VAChT were amplified by PCR (Supplementary Table S1). The amplified promoter regions of MCK and VAChT were inserted into the NotI and HindIII sites of CMV-RSPO2 to substitute for the CMV promoter and to generate MCK-RSPO2 and VAChT-RSPO2, respectively (Fig. 1). Lack of PCR artifacts were confirmed by sequencing the entire inserts.

Lubricin, RSPOs and LGRs were immune stained using
the two-step immunohistochemistry procedure according
to the manufacturer’s protocol (Santa Cruz Biotechnology,
USA). In short, the tissue sections were treated with
rabbit polyclonal antibody for lubricin (Santa Cruz Immunohistochemistry
Lubricin, RSPOs and LGRs were immune stained using
the two-step immunohistochemistry procedure according
to the manufacturer’s protocol (Santa Cruz Biotechnology,
USA). In short, the tissue sections were treated with
rabbit polyclonal antibody for lubricin (Santa Cruz Biotechnology, USA), RSPO1 (Sigma-Aldrich, USA),
RSPO2 (Sigma-Aldrich, USA), LGR5 (Sigma-Aldrich,
USA), LGR6 (Abcam, England) and β-catenin (Santa
Cruz Biotechnology, USA) of 1:500 dilutions at 4˚C.
The slides were washed thrice with phosphate-buffered
saline (PBS, Wel Gene, Korea) and goat anti-rabbit
immunoglobulin G (IgG, Santa Cruz Biotechnology,
USA) was treated at room temperature for 30 minutes.
Western blot bands were developed for visualization with
3, 3′-diaminobenzidine as the chromogen. Each section
was photographed at ×10 magnification by a Zeiss Axio
Cam digital camera.