recombinant fap rfap  (R&D Systems)

Journal: International Journal of Biological Sciences

Article Title: BGN/FAP/STAT3 positive feedback loop mediated mutual interaction between tumor cells and mesothelial cells contributes to peritoneal metastasis of gastric cancer

doi: 10.7150/ijbs.72218

Figure Lengend Snippet: The expression and prognosis of FAP in gastric cancer. * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± SEM (n = 3). a Western blot analysis of GC cells (AGS or HGC27) alone, rFAP-supplemented GC cells, HMrSV5-co-cultured GC cells, CAFLCs-co-cultured GC cells in the presence or absence of stattic. b The relative expression of FAP was detected by qRT-PCR in 65 GC tissues and paired adjacent normal tissues. c - d The association of FAP expression between five-year overall survival (c) and five-year progression-free survival (d) was analyzed by Kaplan-Meier survival analysis. e The correlation of BGN expression with FAP expression in GC tissues. f-g The association of both BGN and FAP expression between five-year overall survival (f) and five-year progression-free survival (g) were analyzed by Kaplan-Meier survival analysis.

Article Snippet: Recombinant human FAP protein (rFAP) (ABclonal, China) was diluted with PBS and utilized at an ultimate concentration of 200 ng/ml.

Techniques: Expressing, Western Blot, Cell Culture, Quantitative RT-PCR

Journal: International Journal of Biological Sciences

Article Title: BGN/FAP/STAT3 positive feedback loop mediated mutual interaction between tumor cells and mesothelial cells contributes to peritoneal metastasis of gastric cancer

doi: 10.7150/ijbs.72218

Figure Lengend Snippet: FAP promotes BGN expression in GC cells by activating STAT3. * P < 0.05, ** P < 0.01, *** P < 0.001. Data are shown as mean ± SEM (n = 3). a Western blot of BGN from GC cells (AGS or HGC27) co-cultured with CAFLCs in a time-dependent manner. b Western blot analysis of GC cells (AGS or HGC27) alone, rFAP-supplemented GC cells, CAFLCs-co-cultured GC cells, and CAFLCs PT100 -co-cultured GC cells for 48h. c The relative expression of BGN was detected in GC cells (AGS or HGC27) alone, rFAP-supplemented GC cells, CAFLCs-co-cultured GC cells for 48h . d The expression of BGN was detected in GC cells (AGS or HGC27) alone, rFAP-supplemented GC cells, CAFLCs-co-cultured GC cells for 48h by ELISA assay. e GC cells (AGS or HGC27) were transfected with a BGN promoter reporter plasmid (pPRO-RB-Report-Basic plasmid) for 24h . GC cells were incubated with or without stattic (10 μM) for 24h before rFAP (200 ng/ml) stimulation, relative luciferase activity of BGN promoter was detected after rFAP stimulation for 24h. f A reporter plasmid for BGN was constructed by cloning BGN promoter region (WT or NC) or identified STAT3 binding site mutants (MUT-1, MUT-2, MUT-3), and the human plasmid expression STAT3 (STAT3-Ex, STAT3-NC) were co-transfected with GC cells (AGS or HGC27), relative luciferase activity of BGN promoter was detected 48h after transfection. g-h GC cells (AGS and HGC27) were stimulated with rFAP. The qRT-PCR of CHIP products demonstrated the direct binding ability of STAT3 to BGN promoter region in GC cells, the input (2%) (g). The qRT-PCR of CHIP products was analyzed including negative control (NC), CHIP1, CHIP2, and CHIP3. The values were normalized to input (2%), and agarose gel (2%) (h).

Article Snippet: Recombinant human FAP protein (rFAP) (ABclonal, China) was diluted with PBS and utilized at an ultimate concentration of 200 ng/ml.

Techniques: Expressing, Western Blot, Cell Culture, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Incubation, Luciferase, Activity Assay, Construct, Clone Assay, Binding Assay, Quantitative RT-PCR, Negative Control, Agarose Gel Electrophoresis

Journal: Bone Research

Article Title: Inhibition of fibroblast activation protein ameliorates cartilage matrix degradation and osteoarthritis progression

doi: 10.1038/s41413-022-00243-8

Figure Lengend Snippet: Fap degrades denatured or MMP13-cleaved Col II. a Fap degrades denatured Col II in a dose-dependent manner. Denatured Col II (boiled at 95 °C for 10 min) was incubated with different amounts of rFap at 37 °C for 24 h. Samples were separated by SDS‒PAGE and quantified by colloidal blue staining ( n = 3 independent experiments). b Fap degrades denatured Col II in a time-dependent manner. Denatured Col II was incubated with rFap at 37 °C for 0–24 h ( n = 3 independent experiments). c FAPi inhibits Fap-mediated degradation of denatured Col II. Different doses of FAPi were preincubated with rFap for 30 min before denatured Col II was added and further incubated at 37 °C for 24 h ( n = 3 independent experiments). d Immunoprecipitation of Fap. HEK293T cells were transfected with GFP control, Oln-Flag, Fap-HA, or Oln-Flag + Fap-HA. Two days after transfection, Fap was immunoprecipitated from total cell lysates with 10 μL anti-HA affinity gel. Five percent total cell lysates were loaded as an input control ( n = 3 independent experiments). e Oln inhibits the Fap-mediated degradation of denatured Col II. Col II was coincubated with immunoprecipitated samples (in 10 μL anti-HA affinity gel) at 37 °C for 24 h ( n = 3 independent experiments). f Fap degrades MMP13-cleaved Col II. Native Col II was preincubated with rFap or rMMP13 at 37 °C for 12 h. EDTA was then added to the reaction mixture with or without rFap and incubated at 37 °C for another 12 h ( n = 3 independent experiments). g Grayscale quantification of the 55, 40 and 30 kDa digestion bands in ( f ). h Experimental design. Primary chondrocytes were stimulated with vehicle control (PBS), 200 ng·mL −1 rFap, 200 ng·mL −1 rMMP13, or 200 ng·mL −1 rFap plus 200 ng·mL −1 rMMP13 for 24 h. i Western blot analysis of mouse Col2a1 protein levels in primary chondrocytes stimulated as in ( h ) ( n = 3 independent experiments). The statistical significance was assessed using one-way ANOVAs with Tukey’s multiple comparison tests. Data are presented as the mean ± SD (* P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: The enzymatic reaction was initiated by adding different doses of rFap (R&D Systems, 8647-SE-010), native or denatured (heated at 95 °C for 10 min) bovine Col II (Chondrex, 20021) into 10 μL PBS buffer and incubated for 1–24 h at 37 °C.

Techniques: Incubation, Staining, Immunoprecipitation, Transfection, Control, Western Blot, Comparison