Ab45039

8 PDP patients and 4 relative normal controls’ paraffin embedded gastric mucosa tissues were performed immunohistochemical staining. Specimens were grilled for 2 hours at 60° C oven at first. Then the specimens were dewaxed with xylene and rehydrated with gradient alcohol. Antigen retrieval was performed by using 0.01 mmol citrate buffer to make the samples infiltrate in for 15 minutes after steaming in pressure-cooker. After retrieval, the tissues were dealt with rabbit antibody against human IL-6 (abcam, ab9324) and mouse antibody against human RANKL (abcam, ab45039) and TNFα (abcam, ab6671) overnight at 4 °C. Biotinylated secondary antibody was adopted in SP kit at room temperature for 10 minutes followed with incubating of an appropriate amount of HRP-conjugated secondary antibody at room temperature for 30 minutes. Immunostaining was carried out through using DAB for chromogen and hematoxylin for nuclear staining. The specimens were cleared in xylene after dehydrated with gradient alcohol. Finally the neutral beams were applied to seal the specimens. All immunostained specimens were examined with the microscope (BA210T, Motic) and assessed by two pathologists blinding to clinical features. Under the Microscopy, the images and IOD (integrated optical density) were acquired with the Image-Pro Plus software 6.0.

Cells were lysed with RIPA buffer mixed with protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Total protein (25 μg) was separated by 10% SDS–polyacrylamide gel and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore). After being blocked in 5% non‐fat milk in Tris‐buffered saline containing 0.1% Tween‐20 for 1 hour at room temperature, the membranes were incubated overnight at 4°C with primary antibodies as following: anti‐TRPV4 (ab39260; Abcam), anti‐Ki67 (ab8191; Abcam), anti‐IL‐6 (ab9324; Abcam), anti‐RANKL (ab45039, Abcam), anti‐osteoprotegerin (OPG) (ab11994, Abcam), anti‐phospho‐p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (4370, Cell Signaling Technology), anti‐p44/42 MAPK (ERK1/2) (4695, Cell Signaling Technology), and anti‐glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) (3683S, Cell Signaling Technology). Then, the membranes were incubated with a horseradish peroxidase‐conjugated mouse or rabbit IgG (1:5000; Zhongshanjinqiao), and protein bands were detected by enhanced with a SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). The relative density of three comparable results was measured using ImageJ software. Each experiment was repeated three times.

We performed immunohistochemical staining experiments with a two-step detection kit (Zhongshan Golden Bridge Biotechnology, Beijing, China) as previously described.^{52 (link)} The primary antibodies used were anti-GLUT1 (1:100, 21829-1-AP, Proteintech), anti-RANKL (1:100, ab45039, Abcam), and anti-phospho-ERK (1:100, 4370, Cell Signaling Technology).

Tissue sections were deparaffinized in xylene, then dehydrated in alcohol and incubated in citric acid solution (MXB Biotechnologies, Fuzhou, China) for 10 min at 90 °C to remove surface antigens. After rinsing with PBS, Cell and Tissue Staining Kits (R&D Systems, MN, USA) were used to detect antigens according to the manufacturer's protocols. Tissue sections were stained overnight at 4 °C with an anti-RANKL antibody (1:100; ab45039, Abcam, Cambridge, UK) or an anti-OPG antibody (1:200; ab9986, Abcam, Cambridge, UK), and after rinsing with PBS, they were incubated for 1 h with a secondary antibody (1:2000; ab97240, Abcam, Cambridge, UK; 1:1000 ab6721, Abcam, Cambridge, UK), a fluorescence microscope system (OLYMPUS, Tokyo, Japan) was used to capture images.

Specimens were incubated overnight at 4°C with primary rabbit polyclonal anti-C/EBPβ antibodies (C-19; Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted 1:100, mouse monoclonal anti-RANKL antibodies (ab45039; Abcam, Cambridge, England) diluted 1:50, gout polyclonal anti-OPG antibodies (sc-8468; Santa Cruz Biotechnology) diluted 1:100, rabbit polyclonal anti-ATF4 antibodies (sc-200; Santa Cruz Biotechnology) or normal rabbit IgG (sc-2027; Santa Cruz Biotechnology) diluted 1:100, respectively. RA-FLS plated on glass coverslips were transfected with adenovirus expression vectors for C/EBPβ-LAP, −LIP or LacZ control [16 (link)] for 24 hours and then replaced with fresh medium. After 48 hours, immunofluorescence staining was performed.