frem1  (Proteintech)

Journal: Bioactive Materials

Article Title: A continuous adhesion-enhanced osteogenic pathway in artificial scaffold drives cellular infiltration and condensed mineralization for rapid bone regeneration

doi: 10.1016/j.bioactmat.2026.02.026

Figure Lengend Snippet: IHC and IF staining of functional proteins. (a) H&E and Masson's staining of defect area after implantation of CPH/rGO-3/0 and CPH/rGO-3/0.6 scaffolds for one month. (b) OCN IHC and IF staining of CPH/rGO-3/0 and CPH/rGO-3/0.6 after implantation for one month and the corresponding OD value and fluorescence intensity statistics. (c) IF staining of FREM1 and ANK3 in scaffolds after implanting for one months in rats and corresponding fluorescence intensity statistics. (d) IF staining of KCNN3 and KCNH1 in scaffolds after implanting for one months in rats and corresponding fluorescence intensity statistics. n = 20 per group. Data are expressed as mean ± SD. ∗ for p < 0.05; ∗∗ for p < 0.01; ∗∗∗ for p < 0.001.

Article Snippet: Hematoxylin-Eosin (Solarbio, Beijing, China) Stain Kit, Masson's Trichrome Stain Kit (Solarbio, Beijing, China), OCN monoclonal antibody (ThermoFisher Scientific, USA) FREM1 and KCNN3 antibody (Bioss Antibodies, Beijing, China), ANK3 antibody (Proteintech, Wuhan, China), KCNH1 (Servicecbio, Wuhan, China) were used to stain the slice of specimens.

Techniques: Staining, Functional Assay, Fluorescence

Journal: Journal of Inflammation Research

Article Title: TILRR (Toll-like Interleukin-1 Receptor Regulator), an Important Modulator of Inflammatory Responsive Genes, is Circulating in the Blood

doi: 10.2147/JIR.S325553

Figure Lengend Snippet: List of Mouse Anti-FREM1 mAbs Selected for TILRR and Full-Length FREM1 Isoform Measurement in Human Plasma Samples

Article Snippet: After the removal of the blocking buffer, the membrane was incubated separately with 20 µg/mL (diluted in PBS-T containing 0.25% BSA [Sigma-Aldrich, Oakville, Canada]) of anti-FREM1 primary mAb (F237G3, F218G4, and F237G1) overnight at 4° C on a shaker (60 rpm).

Techniques: Sequencing

Journal: Journal of Inflammation Research

Article Title: TILRR (Toll-like Interleukin-1 Receptor Regulator), an Important Modulator of Inflammatory Responsive Genes, is Circulating in the Blood

doi: 10.2147/JIR.S325553

Figure Lengend Snippet: TILRR protein detection methods using four mouse anti-FREM1 monoclonal antibodies with multiplex bead array system. F237G3, F218G4, and F237G1 mAbs were used as capture antibodies that coupled with magnetic beads (purple-colored), and F237G12 mAb was used as a detection antibody, which labeled with biotin (red-colored). Both capture and detection antibodies targeted major epitopes on TILRR/FREM1 proteins were formed a sandwich complex that was detected in the Luminex detection system (Bio-Rad, Mississauga, Canada) following the addition of Streptavidin-PE.

Article Snippet: After the removal of the blocking buffer, the membrane was incubated separately with 20 µg/mL (diluted in PBS-T containing 0.25% BSA [Sigma-Aldrich, Oakville, Canada]) of anti-FREM1 primary mAb (F237G3, F218G4, and F237G1) overnight at 4° C on a shaker (60 rpm).

Techniques: Multiplex Assay, Magnetic Beads, Labeling, Luminex

Journal: Journal of Inflammation Research

Article Title: TILRR (Toll-like Interleukin-1 Receptor Regulator), an Important Modulator of Inflammatory Responsive Genes, is Circulating in the Blood

doi: 10.2147/JIR.S325553

Figure Lengend Snippet: Standard curves generated by multiplex bead array using recombinant FREM1 (rFREM1) protein and anti-FREM1 mAbs. rFREM1 spD protein was diluted 5-fold with concentration ranges from 10,000 ng/mL (S1, standard 1) to 0.64 ng/mL (S7, standard 7). Standard curves were generated with three anti-FREM1 mAbs, such as F237G3 (A), F218G4 (B), and F237G1 (C) that coupled with three different fluorescent spectrum beads. As a detection antibody, custom biotinylated anti-FREM1 (F237G12) mAb was used. MFI value for each antibody was graphed against the corresponding observed concentration of each point of rFREM1 spD protein (S1 to S7) where bead recovery ranges were maintained 70–130. Each line represents one replicate. A total of three independent experiments (n=3) were conducted for each antibody (all three antibodies were tested together as a 3-plex). The X-axis indicates the protein concentration (ng/mL) and Y-axis represents the MFI.

Article Snippet: After the removal of the blocking buffer, the membrane was incubated separately with 20 µg/mL (diluted in PBS-T containing 0.25% BSA [Sigma-Aldrich, Oakville, Canada]) of anti-FREM1 primary mAb (F237G3, F218G4, and F237G1) overnight at 4° C on a shaker (60 rpm).

Techniques: Generated, Multiplex Assay, Recombinant, Concentration Assay, Protein Concentration

Journal: Journal of Inflammation Research

Article Title: TILRR (Toll-like Interleukin-1 Receptor Regulator), an Important Modulator of Inflammatory Responsive Genes, is Circulating in the Blood

doi: 10.2147/JIR.S325553

Figure Lengend Snippet: TILRR Protein Detection and Quantification in Human Blood Plasma Using the Multiplex Bead Array Method

Article Snippet: After the removal of the blocking buffer, the membrane was incubated separately with 20 µg/mL (diluted in PBS-T containing 0.25% BSA [Sigma-Aldrich, Oakville, Canada]) of anti-FREM1 primary mAb (F237G3, F218G4, and F237G1) overnight at 4° C on a shaker (60 rpm).

Techniques: Multiplex Assay

Journal: Journal of Inflammation Research

Article Title: TILRR (Toll-like Interleukin-1 Receptor Regulator), an Important Modulator of Inflammatory Responsive Genes, is Circulating in the Blood

doi: 10.2147/JIR.S325553

Figure Lengend Snippet: Western blot and Coomassie blue staining confirmation of purified TILRR protein from human blood plasma after affinity purification. The tables on the top of the figures show the amount of TILRR protein quantified by multiplex bead array using plasma samples from groups 1–4 . (A–D) Affinity purified TILRR protein from human blood plasma was analyzed by Western blot (left, green color) and Coomassie blue staining of NuPAGE gel after iBlot transfer, showing the untransferred protein (right, red color) in Group 1 (A), Group 2 (B), Group 3 (C), and Group 4 (D). Each group shows data from 3 patients (n=3). Top panels (left) of figures A-D show the Western blot bands (green color) for TILRR protein (cropped) detected by two different anti-FREM1 IgG mAbs (F237G3 and F218G4) (full-length un-cropped blot images are shown in Figure S4 ). Top panels (right) of figures A-D show the protein bands in the gel after Coomassie blue staining (red color) (cropped); Full-length un-cropped gels are shown in Figure S5 . The corresponding TILRR protein (R.F.U) for each patient is shown in the bar graph beneath the cropped blots and gels. Western blot and Coomassie blue staining images were acquired by Odyssey CLx imaging system (LI-COR, USA) with auto channels (both 700 and 800), 42 µm resolution, high image quality, and 0 mm focal offset for blots and 0.5 mm focal offset for gels. The X-axis of all figures indicates the patients’ identification number and Y-axis shows the TILRR protein (R.F.U).

Article Snippet: After the removal of the blocking buffer, the membrane was incubated separately with 20 µg/mL (diluted in PBS-T containing 0.25% BSA [Sigma-Aldrich, Oakville, Canada]) of anti-FREM1 primary mAb (F237G3, F218G4, and F237G1) overnight at 4° C on a shaker (60 rpm).

Techniques: Western Blot, Staining, Purification, Affinity Purification, Multiplex Assay, Imaging

Journal: Journal of Inflammation Research

Article Title: TILRR (Toll-like Interleukin-1 Receptor Regulator), an Important Modulator of Inflammatory Responsive Genes, is Circulating in the Blood

doi: 10.2147/JIR.S325553

Figure Lengend Snippet: TILRR Protein Detection and Confirmation in Human Blood Plasma by the Multiplex Bead Array Method and Western Blot Analysis

Article Snippet: After the removal of the blocking buffer, the membrane was incubated separately with 20 µg/mL (diluted in PBS-T containing 0.25% BSA [Sigma-Aldrich, Oakville, Canada]) of anti-FREM1 primary mAb (F237G3, F218G4, and F237G1) overnight at 4° C on a shaker (60 rpm).

Techniques: Multiplex Assay, Western Blot

Journal: Journal of Inflammation Research

Article Title: TILRR (Toll-like Interleukin-1 Receptor Regulator), an Important Modulator of Inflammatory Responsive Genes, is Circulating in the Blood

doi: 10.2147/JIR.S325553

Figure Lengend Snippet: TILRR Protein Detection and Quantification in Human Blood Plasma by the Multiplex Bead Array Method (After Western Blot Analysis)

Article Snippet: After the removal of the blocking buffer, the membrane was incubated separately with 20 µg/mL (diluted in PBS-T containing 0.25% BSA [Sigma-Aldrich, Oakville, Canada]) of anti-FREM1 primary mAb (F237G3, F218G4, and F237G1) overnight at 4° C on a shaker (60 rpm).

Techniques: Multiplex Assay, Western Blot

Journal: Journal of Inflammation Research

Article Title: TILRR (Toll-like Interleukin-1 Receptor Regulator), an Important Modulator of Inflammatory Responsive Genes, is Circulating in the Blood

doi: 10.2147/JIR.S325553

Figure Lengend Snippet: Variable quantity and the detection frequency of TILRR protein in human plasma samples. (A) The level of TILRR protein detected by anti-FREM1 mAbs (F237G3 and F218G4) among plasma groups 1–4 (n=640) (as classified in ) is shown (median with interquartile range [IQR]). In the case of group 1 (n=186), both anti-FREM1 mAbs (F237G3 and F218G4) detected the TILRR protein in blood plasma, thus the higher detection value between these two mAbs was plotted (plasma group 1). In the case of groups 2 (n=303) and 3 (n=19), TILRR protein was only detected by F237G3 mAb (group 2) and F218G4 mab (group 3), respectively. TILRR was detected neither by F237G3 mAb nor by F218G4 mAb in group 4 (below the detection limit, n=132). Thus, the data for group 4 was plotted as 0 (zero). F237G3 mAb targets epitopes on CSPG11 and 12 domains of FREM1/TILRR protein. F218G4 mAb binds epitopes on the Calx-β domain of FREM1/TILRR protein. (B) The detection frequency of TILRR protein among plasma groups 1–4 (n=640). As plasma group 4 (n=132) did not show TILRR protein detection by multiplex bead array method, the detection frequency for group 4 showed as 0 (zero)%. For groups 1–3, the detection frequency was calculated based on the total detected samples (n=508; group 1 [n=186], group 2 [n=303] and group 3 [n=19]). The sample dilution buffer was used as a negative control. Since the control buffer did not provide any data (below the lower limit of detection), the negative control condition is not shown in the figures. Statistical comparisons were conducted using a Student’s t -test with 95% CI (confidence intervals), all p<0.05 were reported and indicated using asterisks’ ****p<0.0001. The X-axis of both figures A and B show the plasma groups, whereas the Y-axis of figures A and B indicates the TILRR protein concentration (ng/mL) and detection frequency (%), respectively.

Article Snippet: After the removal of the blocking buffer, the membrane was incubated separately with 20 µg/mL (diluted in PBS-T containing 0.25% BSA [Sigma-Aldrich, Oakville, Canada]) of anti-FREM1 primary mAb (F237G3, F218G4, and F237G1) overnight at 4° C on a shaker (60 rpm).

Techniques: Multiplex Assay, Negative Control, Protein Concentration

Journal: Journal of Inflammation Research

Article Title: TILRR (Toll-like Interleukin-1 Receptor Regulator), an Important Modulator of Inflammatory Responsive Genes, is Circulating in the Blood

doi: 10.2147/JIR.S325553

Figure Lengend Snippet: Western blot and Coomassie blue confirmation of TILRR protein in plasma samples spiked with rFREM1 spD and following affinity purification. The plasma sample (1 mL) was spiked with rFREM1 spD (106.58 ng/mL) in a single tube. In parallel, the plasma sample alone, and rFREM1 spD alone were separately taken into the tubes. The final volume of all samples was adjusted to 4 mL (1:4 dilution), and the concentration of spiked rFREM1 spD protein was adjusted similar to the concentration of TILRR protein (median) in the patient’s plasma observed by the multiplex bead array method. Affinity purification (AP) was conducted by cross-linked anti-FREM1 F237G12 IgG mAb. (A) Western blot confirmation of ~57 kDa rFREM1 spD protein (cropped; Full-length un-cropped bot is shown in Figure S7A ). The bar graph underneath the cropped blot shows the corresponding protein intensity (R.F.U) for only ~57 kDa protein. (B) Coomassie blue staining of ~57 kDa rFREM1 spD protein (cropped; the full-length un-cropped gel is shown in Figure S7B ). The bar graph underneath the cropped gel shows the corresponding rFREM1spD protein intensity (R.F.U) for ~57 kDa protein. TILRR protein intensity (R.F.U) with bar graph for 70 kDa protein is not shown. C-D) Plasma samples with spiked rFREM1 spD protein (R.F.U) compared to the rFREM1 spD alone and plasma alone following Western blot analysis (C) and Coomassie blue staining (D) (n=2). Western blot and Coomassie blue staining images were acquired by Odyssey CLx imaging system (LI-COR, USA) with auto channels (both 700 and 800), 42 µm resolution, high image quality, and 0 mm focal offset for blots and 0.5 mm focal offset for gels.

Article Snippet: After the removal of the blocking buffer, the membrane was incubated separately with 20 µg/mL (diluted in PBS-T containing 0.25% BSA [Sigma-Aldrich, Oakville, Canada]) of anti-FREM1 primary mAb (F237G3, F218G4, and F237G1) overnight at 4° C on a shaker (60 rpm).

Techniques: Western Blot, Affinity Purification, Concentration Assay, Multiplex Assay, Staining, Imaging

Journal: Human Molecular Genetics

Article Title: A homozygous mutation p.Arg2167Trp in FREM2 causes isolated cryptophthalmos

doi: 10.1093/hmg/ddy144

Figure Lengend Snippet: Function of FREM2 mutation p.Arg2167Trp. (A) Cellular localization of FREM2–WT and FREM2–Arg2167Trp mutant in HEK293T treated with the siRNA and reintroduced it by transfecting siRNA-resistant plasmids: pFREM2–WT or pFREM2–Arg2167Trp. Cells were stained with anti-FREM2 antibody (S-12, Santa Cruz). Nuclei was stained by DAPI (Blue). (B) Growth curve analysis of HEK293T cells transfected with either FREM2–WT or FREM2–Arg2167Trp. Control was done in cells transfected with pcDNA3.1. Error bars represent SEM of six independent experiments. (C) Fibronectin adhesion assay of HEK293T treated with WT or mutant FREM2 as described in Materials and Methods. Error bars represent SEM of six independent experiments. (D) Silver stained and immunoblotted SDS-PAGE gel. Lane 1 is ladder; Lanes 2–5 are immunoprecipitated FREM2 using monoclonal antibody anti-FREM2 (F-1, Santa Cruz); Lanes 6 and 7 immunoprecipitated FREM1 using anti-FREM1 (H-300), solid arrow indicates FREM1 and open arrow indicates FREM2, which is confirmed by immunoblotting with anti-FREM1 (H-300, Santa Cruz) and FREM2 (S-18, Santa Cruz) antibodies (bottom panel). Quantification of the band's density was performed using ImageJ. The capacity of binding of FREM1 was calculate using formulas: Capacity=FREM1Lane nFREM2Lane n. The capacity of binding of FREM1 in Lane 2 was set as 1, and the number under Lanes 3 and 4 indicates the relative capacity of the mutant FREM2 as compared with WT (Lane 2). RD, relative capacity of coimmunoprecipitated FREM1.

Article Snippet: The primary antibodies included rabbit anti-FREM2 (1:300, S-18, Santa Cruz), rabbit anti-FREM1 (1:300, H-300, Santa Cruz) and rabbit anti-GAPDH (1:1000, CST).

Techniques: Mutagenesis, Staining, Transfection, Cell Adhesion Assay, SDS Page, Immunoprecipitation, Western Blot, Binding Assay

Journal: Human Molecular Genetics

Article Title: A homozygous mutation p.Arg2167Trp in FREM2 causes isolated cryptophthalmos

doi: 10.1093/hmg/ddy144

Figure Lengend Snippet: Function of FREM2 mutation p.Arg2167Trp. (A) Cellular localization of FREM2–WT and FREM2–Arg2167Trp mutant in HEK293T treated with the siRNA and reintroduced it by transfecting siRNA-resistant plasmids: pFREM2–WT or pFREM2–Arg2167Trp. Cells were stained with anti-FREM2 antibody (S-12, Santa Cruz). Nuclei was stained by DAPI (Blue). (B) Growth curve analysis of HEK293T cells transfected with either FREM2–WT or FREM2–Arg2167Trp. Control was done in cells transfected with pcDNA3.1. Error bars represent SEM of six independent experiments. (C) Fibronectin adhesion assay of HEK293T treated with WT or mutant FREM2 as described in Materials and Methods. Error bars represent SEM of six independent experiments. (D) Silver stained and immunoblotted SDS-PAGE gel. Lane 1 is ladder; Lanes 2–5 are immunoprecipitated FREM2 using monoclonal antibody anti-FREM2 (F-1, Santa Cruz); Lanes 6 and 7 immunoprecipitated FREM1 using anti-FREM1 (H-300), solid arrow indicates FREM1 and open arrow indicates FREM2, which is confirmed by immunoblotting with anti-FREM1 (H-300, Santa Cruz) and FREM2 (S-18, Santa Cruz) antibodies (bottom panel). Quantification of the band's density was performed using ImageJ. The capacity of binding of FREM1 was calculate using formulas: Capacity=FREM1Lane nFREM2Lane n. The capacity of binding of FREM1 in Lane 2 was set as 1, and the number under Lanes 3 and 4 indicates the relative capacity of the mutant FREM2 as compared with WT (Lane 2). RD, relative capacity of coimmunoprecipitated FREM1.

Article Snippet: Bead-bound proteins were resolved by 7.5% SDS-PAGE and examined by silver staining and subsequent immunoblotting with anti-FREM1 (1:300, H-300, Santa Cruz) and FREM2 (1:300, S-18, Santa Cruz) antibodies.

Techniques: Mutagenesis, Staining, Transfection, Cell Adhesion Assay, SDS Page, Immunoprecipitation, Western Blot, Binding Assay