anti opg antibody  (R&D Systems)

Journal: JBMR Plus

Article Title: Osteocytes regulate osteoprotegerin expression via the p38-MAPK-CREB pathway in rheumatoid arthritis

doi: 10.1093/jbmrpl/ziag023

Figure Lengend Snippet: TNF-α most strongly modulates osteocyte-related gene and protein expression. (A) Dot plots of the relative mRNA levels for Tnfsf11 (RANKL) and Tnfrsf11b (OPG) in MLO-Y4 cells stimulated with TNF-α, IL-6 or IL-17 ( n = 3). (B) Dot plots showing enrichment of the osteocyte marker Sost in osteocyte-enriched bone fraction (OEBFs) vs non-digested bone ( n = 3). (C) Dot plots of Tnfsf11 , Tnfrsf11b , and Sost mRNA in OEBF with or without TNF-α ( n = 3). (D) Representative immunoblot of OPG and Sclerostin in OEBF with or without TNF-α; β-actin serves as the loading control. (E) Dot plots of secreted OPG in conditioned medium from MLO-Y4 cells treated with TNF-α, measured by ELISA ( n = 3). Data in (A-C and E) are individual values with the mean ± SD; * p < .05, ** p < .01, *** p < .001; ns indicates not significant by the 2-sided Student’s t -test.

Article Snippet: For osteoclast differentiation assays, the following 5 conditions were compared (all containing 100 ng/mL RANKL): (1) no MLO-Y4 CM and no TNF-α (RANKL only; no TNF source); (2) TNF-α added directly to osteoclast cultures (TNF-α, no MLO-Y4); (3) Unstim CM added at 10% (v/v); (4) TNF-stim CM added at 10% (v/v); and (5) TNF-stim CM pre-incubated with a neutralizing anti-OPG antibody (AF459, R&D Systems) prior to addition to osteoclast cultures.

Techniques: Expressing, Marker, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: JBMR Plus

Article Title: Osteocytes regulate osteoprotegerin expression via the p38-MAPK-CREB pathway in rheumatoid arthritis

doi: 10.1093/jbmrpl/ziag023

Figure Lengend Snippet: TNF-α-induced changes in Wnt/β-catenin-related transcripts and activation of the p38-MAPK-CREB signaling pathway in osteocytes. (A) Dot plots showing relative mRNA levels of Ctnnb1 (β-catenin), Lef1 , and Axin2 in MLO-Y4 cells treated with or without TNF-α ( n = 3). (B) Representative immunoblotting images showing phosphorylated p38 MAPK (p-p38), total p38, phosphorylated CREB (p-CREB), total CREB, phosphorylated JNK (p-JNK), total JNK, phosphorylated ERK (p-ERK), total ERK, and β-actin in MLO-Y4 cells following TNF-α treatment. Data in (A) are shown as individual values with the mean ± SD and were analyzed by the two-sided Student’s t -test. (C) Representative immunoblot of OPG in TNF-α-stimulated MLO-Y4 cells treated with a p38 inhibitor or a CREB inhibitor. Cells were stimulated with TNF-α in the presence or absence of SB203580 (p38 inhibitor) or 666-15 (CREB inhibitor), and total cell lysates were subjected to immunoblotting for OPG. β-actin serves as the loading control. Numeric values shown beneath each band represent the relative expression levels of OPG/β-actin for the corresponding lane. (D) ChIP-PCR demonstrating enrichment of the OPG promoter in anti-CREB immunoprecipitates from MLO-Y4 cells with or without TNF-α stimulation, with densitometric quantification of the ChIP-PCR bands; input DNA served as the internal control, normal IgG as the negative control, and anti-histone H3 as the positive control.

Article Snippet: For osteoclast differentiation assays, the following 5 conditions were compared (all containing 100 ng/mL RANKL): (1) no MLO-Y4 CM and no TNF-α (RANKL only; no TNF source); (2) TNF-α added directly to osteoclast cultures (TNF-α, no MLO-Y4); (3) Unstim CM added at 10% (v/v); (4) TNF-stim CM added at 10% (v/v); and (5) TNF-stim CM pre-incubated with a neutralizing anti-OPG antibody (AF459, R&D Systems) prior to addition to osteoclast cultures.

Techniques: Activation Assay, Western Blot, Control, Expressing, Negative Control, Positive Control

Journal: JBMR Plus

Article Title: Osteocytes regulate osteoprotegerin expression via the p38-MAPK-CREB pathway in rheumatoid arthritis

doi: 10.1093/jbmrpl/ziag023

Figure Lengend Snippet: TNF-α-stimulated osteocyte-conditioned medium (CM) suppresses osteoclast differentiation partially via OPG. (A) Representative TRAP staining of osteoclasts generated under 5 conditions: (1) RANKL alone without any TNF-α source or osteocyte-CM (MLO-Y4−, TNF-α−); (2) direct TNF-α treatment in the absence of MLO-Y4 conditioned medium (MLO-Y4−, TNF-α+); (3) conditioned medium (CM) from unstimulated MLO-Y4 cells (MLO-Y4+, TNF-α−); (4) CM from TNF-α-stimulated MLO-Y4 cells collected after PBS wash and medium replacement (MLO-Y4+, TNF-α+); and (5) TNF-α-stimulated MLO-Y4 CM pre-incubated with a neutralizing anti-OPG antibody (AF459). Scale bars are 500 μm for low magnification and 200 μm for high magnification. (B) Dot plots of the percentage of spreading TRAP-positive osteoclasts for the conditions shown in (A) ( n = 3). (C) Quantitative RT-PCR analysis of Acp5 mRNA expression in osteoclast cultures under the conditions shown in (A). Acp5 expression levels were normalized to the reference gene ( n = 3). Data in (B and C) are individual values with mean ± SD; ** p < .01, **** p < .0001; ns indicates not significant by the one-way ANOVA with Tukey’s post-hoc test.

Article Snippet: For osteoclast differentiation assays, the following 5 conditions were compared (all containing 100 ng/mL RANKL): (1) no MLO-Y4 CM and no TNF-α (RANKL only; no TNF source); (2) TNF-α added directly to osteoclast cultures (TNF-α, no MLO-Y4); (3) Unstim CM added at 10% (v/v); (4) TNF-stim CM added at 10% (v/v); and (5) TNF-stim CM pre-incubated with a neutralizing anti-OPG antibody (AF459, R&D Systems) prior to addition to osteoclast cultures.

Techniques: Staining, Generated, Incubation, Quantitative RT-PCR, Expressing

Journal: BMC Oral Health

Article Title: Chronic intermittent hypobaric hypoxia attenuates root resorption following delayed tooth replantation by upregulating OPG/RANKL signaling pathway

doi: 10.1186/s12903-026-08135-7

Figure Lengend Snippet: CIHH downregulates RANKL expression and increases the OPG/RANKL ratio in delayed replanted teeth. A Immunohistochemistry staining showing OPG expression in the root and periapical tissue of NN and CIHH groups of delayed replanted teeth. Scale bar: 100 μm. B Immunohistochemistry staining showing RANKL expression in the root and periapical tissue of the NN and CIHH groups. Scale bar: 100 μm. C - D Representative Western blot bands ( C ) and quantitative analysis ( D ) showing OPG and RANKL protein expressions in the root and periapical tissue of NN and CIHH groups of delayed replanted teeth. D dentin; P: periodontal ligament; B: bone

Article Snippet: Membranes were blocked with 5% skim milk and incubated with a mouse anti-OPG primary antibody (1:1000, Huabio, China) or a rabbit anti-RANKL primary antibody (1:1000, Huabio, China) overnight at 4 °C.

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot

Journal: BMC Oral Health

Article Title: Chronic intermittent hypobaric hypoxia attenuates root resorption following delayed tooth replantation by upregulating OPG/RANKL signaling pathway

doi: 10.1186/s12903-026-08135-7

Figure Lengend Snippet: CIHH downregulates RANKL expression and increases the OPG/RANKL ratio in delayed replanted teeth. A Immunohistochemistry staining showing OPG expression in the root and periapical tissue of NN and CIHH groups of delayed replanted teeth. Scale bar: 100 μm. B Immunohistochemistry staining showing RANKL expression in the root and periapical tissue of the NN and CIHH groups. Scale bar: 100 μm. C - D Representative Western blot bands ( C ) and quantitative analysis ( D ) showing OPG and RANKL protein expressions in the root and periapical tissue of NN and CIHH groups of delayed replanted teeth. D dentin; P: periodontal ligament; B: bone

Article Snippet: The sections were then incubated overnight at 4 °C with primary antibodies: a mouse anti-OPG antibody (1:1000, Huabio, China) or a rabbit anti-RANKL antibody (1:1000, Huabio, China).

Techniques: Expressing, Immunohistochemistry, Staining, Western Blot

Journal: Immunity, Inflammation and Disease

Article Title: RANKL Attenuates Sepsis‐Associated Acute Lung Injury Through the OPG/RANKL/RANK/TLR4 Pathway

doi: 10.1002/iid3.70356

Figure Lengend Snippet: Comparison of mRNA expression levels of TLR4, RANK, RANKL, and OPG in lung tissues of mice in each group. The mRNA levels were detected with real‐time quantitative PCR. (A) Level of RANKL mRNA. (B) Level of OPG mRNA. (C) Level of RANK mRNA. (D) Level of TLR4 mRNA. *** p < 0.001, **** p < 0.0001.

Article Snippet: After that, the pre‐diluted primary antibodies against OPG (cat# DF6824; Affinity Bioscience), RANKL (cat# 23408‐1‐AP; Proteintech Group Inc.), RANK (cat# ab222215; Abcam), TLR4 (cat# 19811‐1‐AP; Proteintech Group Inc.), and β‐actin (cat# TDY051; Tiandeyue Biotechnology Co. Ltd) were added and probed overnight at 4°C.

Techniques: Comparison, Expressing, Real-time Polymerase Chain Reaction

Journal: Immunity, Inflammation and Disease

Article Title: RANKL Attenuates Sepsis‐Associated Acute Lung Injury Through the OPG/RANKL/RANK/TLR4 Pathway

doi: 10.1002/iid3.70356

Figure Lengend Snippet: Comparison of protein expression levels of TLR4, RANK, RANKL, and OPG in lung tissues of mice in each group. The protein levels were detected with Western blot analysis. (A) Representative Western blot results. (B) The relative protein level of RANKL. (C) The relative protein level of OPG. (D) The relative protein level of RANK. (E) The relative protein level of TLR4. * p < 0.05, **** p < 0.0001.

Article Snippet: After that, the pre‐diluted primary antibodies against OPG (cat# DF6824; Affinity Bioscience), RANKL (cat# 23408‐1‐AP; Proteintech Group Inc.), RANK (cat# ab222215; Abcam), TLR4 (cat# 19811‐1‐AP; Proteintech Group Inc.), and β‐actin (cat# TDY051; Tiandeyue Biotechnology Co. Ltd) were added and probed overnight at 4°C.

Techniques: Comparison, Expressing, Western Blot