primary antibody against fgf8  (Proteintech)

Journal: Scientific Reports

Article Title: Extracellular vesicles derived from Msh homeobox 1 ( Msx1 )-overexpressing mesenchymal stem cells improve digit tip regeneration in an amputee mice model

doi: 10.1038/s41598-024-72647-x

Figure Lengend Snippet: Schematic representation of study design. ( A ) BlCs culture and Msx1 gene expression and characterization. ( B,C ) BlCs-EV isolation and characterization. ( D ) Creation of a non-regenerative digit tip. ( E ) BlCs-EV injection 4 DPA into the models. ( F ) The analysis of digit tip regeneration during 6 WPI. 4 DPA: 4 days post-amputation, 6 WPI: 6 weeks post-implantation.

Article Snippet: Then incubated overnight with primary antibodies against BMP4, FGF8, MSX1, and MSX2 proteins (Invitrogen) at 4 °C.

Techniques: Expressing, Isolation, Injection

Journal: Scientific Reports

Article Title: Extracellular vesicles derived from Msh homeobox 1 ( Msx1 )-overexpressing mesenchymal stem cells improve digit tip regeneration in an amputee mice model

doi: 10.1038/s41598-024-72647-x

Figure Lengend Snippet: BlCs culture, expansion, and Msx1 , Msx2 , Fgf8 , and Bmp4-related gene expression. ( A ) Fluorescent microscopic images of Msx1 (GFP + ) show Msx1 expression in BlCs immediately after culture and expansion (a), the nucleus of cells stained with DAPI dye (blue) (b), and illustrated the merged of a and b images ( n = 3) (c). ( B ) Real-time PCR analysis shows the Msx1 (a), Msx2 (b), Bmp4 (c), and Fgf8 (d) gene expression of BlCs in comparison with mBMSCs as a control group ( n = 3). Scale bar: 100 μm. Data are presented as means ± SD, (**** p < 0.001).

Article Snippet: Then incubated overnight with primary antibodies against BMP4, FGF8, MSX1, and MSX2 proteins (Invitrogen) at 4 °C.

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

Journal: Scientific Reports

Article Title: Extracellular vesicles derived from Msh homeobox 1 ( Msx1 )-overexpressing mesenchymal stem cells improve digit tip regeneration in an amputee mice model

doi: 10.1038/s41598-024-72647-x

Figure Lengend Snippet: EVs isolation and characterization. ( A ) WB assessment indicated that the BlCs-EV were positive for CD81, a main marker of EVs. ( B ) Size and morphology of EVs obtained by SEM images, ( C ) Size of EVs observed using DLS (approximately 100–150 nm. ( D ) WB analysis of the EVs enriched proteins (BMP4, FGF8, and MSX1, MSX1). As full as possible length blots are presented in additional file 1: Figure Supplementary . The quantitative expression of the proteins MSX1, MSX2, FGF8, and BMP4 in BlCs-EVs and mBMSCs-EVs group ( n = 1). EVs: extracellular vesicle; WB: Western Blot; DLS: SEM: Scanning electron microscopy.

Article Snippet: Then incubated overnight with primary antibodies against BMP4, FGF8, MSX1, and MSX2 proteins (Invitrogen) at 4 °C.

Techniques: Isolation, Marker, Expressing, Western Blot, Electron Microscopy

Journal: OncoImmunology

Article Title: Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

doi: 10.1080/2162402x.2022.2096349

Figure Lengend Snippet: Figure 1. FGF8 autoantibodies detection in very high-risk ARMS patients. (a) Left, experimental workflow followed for the identification of autoantibodies in ARMS patients using plasma samples and ProtoArrayTM technology.31 The immune response profile was obtained from 10 metastatic ARMS patients and 15 healthy subjects (HS), probing protein microarray chips with plasma. Reactivity of 9374 spotted antigens was evaluated after signal detection, filtration and normalization using robust linear model (RLM). Antigens median values were calculated for each group, compared and ranked according to significant p-value scale. Right, volcano plot of protein microarray data showing differentially immunoreactive antigens between patients and healthy subjects, plotted along dimension of fold change (abscissae) and statistical difference (ordinates). Antigens with significant p-values (≥0,05) are indicated by colors and names, while antigens with no significant difference in immunoreactivity between patients and controls are plotted uncolored on the bottom of the graph (gray dots). Antigens more reactive in patients or controls are distinguished by red or green dots, respectively. (b) Venn diagram showing the overlap between differential immunoreactive antigens (n = 55) and PAX3-FOXO1 target genes (n = 1010).32 (c) Box plot of FGF8 signal intensity revealed in patients and controls by protein microarray (RFU = relative fluorescence unit) and (d) validation by indirect ELISA assay. (e) Correlation of FGF8 autoantibody signal intensity and FGF8 IgG concentration obtained in the same samples cohort by protein microarrays and ELISA assay, respectively. p < 0,05 (*); p < 0,01 (**).

Article Snippet: Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes.

Techniques: Clinical Proteomics, Microarray, Filtration, Fluorescence, Biomarker Discovery, Indirect ELISA, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: OncoImmunology

Article Title: Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

doi: 10.1080/2162402x.2022.2096349

Figure Lengend Snippet: Figure 2. Correlation between humoral immune response against FGF8 and patients’ outcome. Kaplan-Meier survival analysis representing (a) event-free survival (EFS) and (b) overall survival (OS) of ARMS patients distinguished accord ing to FGF8 autoantibodies median value.

Article Snippet: Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes.

Techniques:

Journal: OncoImmunology

Article Title: Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

doi: 10.1080/2162402x.2022.2096349

Figure Lengend Snippet: Figure 3. Expression of FGF8 in RMS cell lines. (a) Relative quantification of FGF8 mRNA by qRT-PCR in normal control cells (CTR, n = 4), alveolar RMS (ARMS, n = 5), embryonal RMS (ERMS, n = 5), Ewing sarcoma (EWS, n = 7), Non-Hodgkin lymphoma cell lines (NHL, n = 5), leukemia cell lines (Leukemia, n = 3) and cell lines of various origins (Others, n = 10). Statistical significance was calculated by Mann-Whitney U test, comparing each group of cell lines with ARMS group. Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) housekeeping gene was used for normalization, while normal skeletal muscle tissue extracts were used as external calibrator. (b) FGF8 protein expression and localization by immunocytochemistry and (c) immunofluorescence analysis in RH30 (PF+ ARMS) and RD (PF− ERMS) cell lines (magnifications of selected areas are shown apart). (d) Western blotting and (e) direct ELISA assay performed using serum-starved RH30 and RD total lysate and growth medium, respectively, to assess FGF8 protein at intracellular and at secreted level. A_SKM, adult skeletal muscle; p < 0,05 (*); p < 0,01 (**).

Article Snippet: Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes.

Techniques: Expressing, Quantitative Proteomics, Quantitative RT-PCR, Control, MANN-WHITNEY, Immunocytochemistry, Immunofluorescence, Western Blot, Direct ELISA

Journal: OncoImmunology

Article Title: Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

doi: 10.1080/2162402x.2022.2096349

Figure Lengend Snippet: Figure 4. FGF8 signaling in RMS cells. (a) Relative quantification of FGFR1-4 receptors mRNA in ARMS and ERMS cell lines. FGF8 mRNA levels are also displayed in graph (red open dots). Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) was used for signal intensity normalization, while skeletal muscle extracts were used as external calibrator. (b) FGFR2 and FGFR4 expression and phosphorylation in PF+ (RH30, RH4) and PF− (RD) cell lines, performed through immunoprecipitation of total FGFR2 and FGFR4 receptor proteins. GAPDH protein was used as gel-loading control. (c) Western blotting showing time-dependent phosphorylation of FRS2 and ERK1/2 proteins induced in RH4 and RD cells upon exposure to 50 ng/mL human recombinant FGF8 protein for the indicated time periods. Between blots a graph displaying phosphorylated FRS2 and ERK1/2 band densities, quantified using ImageJ software, was included. γ-Tubulin was used as gel loading control. (d) RH4 and RD cell lines wound healing assay performed in presence and absence of 100 ng/ml human FGF8. Images were taken up to 48 hours after the treatment. (e) Immunoblot analysis of phosphorylated ERK1/2 kinase in RMS cells exposed to increasing concentration of human FGF8 (25, 100 ng/mL), pretreated or not for 2 hours with 5 μM of NVP-BGJ398. γ-Tubulin was used as gel loading control. (f) MTT assay showing PF+ ARMS (RH30, RH4) and PF− ERMS (RH36, RD) cell viability in the presence of 5 μM of NVP-BGJ398 up to 72 hours.

Article Snippet: Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes.

Techniques: Quantitative Proteomics, Expressing, Phospho-proteomics, Immunoprecipitation, Control, Western Blot, Recombinant, Software, Wound Healing Assay, Concentration Assay, MTT Assay

Journal: OncoImmunology

Article Title: Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

doi: 10.1080/2162402x.2022.2096349

Figure Lengend Snippet: Figure 5. FGF8-induced gene expression in RMS cells. (a) Time-dependent expression of DUSP6, SPRY4, GDF15 and ETV5 FGF-target genes, upon treatment of RH4 and RD cells with 100 ng/ml of human recombinant FGF8. Glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) housekeeping gene was used for normalization. (b) STRING analysis. (c) Time-dependent expression of PLAU and MMP-9 genes after treatment of RH4 and RD cell lines with 100 ng/ml of human recombinant FGF8.

Article Snippet: Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes.

Techniques: Gene Expression, Expressing, Recombinant

Journal: OncoImmunology

Article Title: Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

doi: 10.1080/2162402x.2022.2096349

Figure Lengend Snippet: Figure 6. FGF8 expression in RMS tumor biopsies. (a) Relative quantification of FGF8 mRNA in RMS primary tumors (n = 50) and normal controls (n = 4), and (b) in RMS primary tumors divided according to fusion status. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used for normalization. (c) Hematoxylin/Eosin (HE) and FGF8 staining of representative ARMS and ERMS cases. (d) Scatter plot showing the correlation between FGF8 mRNA levels and autoantibodies titer in 33 PF+ ARMS primary tumors and plasma samples, respectively. Vertical and horizontal dashed lines represent FGF8 mRNA and autoantibody median values, respectively, used to divide the plot in four regions (I–IV). Dots represent patients, labeled with different colors based on event occurrence (gray) or not (red) after frontline chemotherapy. p < 0,001 (***); p < 0,0001 (****).

Article Snippet: Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes.

Techniques: Expressing, Quantitative Proteomics, Staining, Clinical Proteomics, Labeling

Journal: OncoImmunology

Article Title: Immunoreactivity against fibroblast growth factor 8 in alveolar rhabdomyosarcoma patients and its involvement in tumor aggressiveness

doi: 10.1080/2162402x.2022.2096349

Figure Lengend Snippet: Figure 7. FGF8 expression in RMS recurrent tumors. (a) FGF8 and PAX3-FOXO1 or PAX7-FOXO1, mRNA at diagnosis and at relapse in 7 cases of ARMS, and (b) 5 ERMS cases. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used for normalization. n.s., not significant; p < 0,05 (*).

Article Snippet: Primary antibody against FGF8 (Proteintech) and in 1%BSA/PBS1X were probed at 37°C for 60 minutes, followed by secondary antibody Alexa Fluor® 546 conjugate (Thermo Fisher scientific) in PBS1X at 37°C for 60 minutes.

Techniques: Expressing, Biomarker Discovery