Hepatocyte growth factor (hgf)

Manufactured by Santa Cruz Biotechnology

Sourced in United States

HGF is a recombinant protein that functions as a hepatocyte growth factor. It is a crucial regulator of cell growth, motility, and morphogenesis.

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Lab products found in correlation

13 protocols using hepatocyte growth factor (hgf)

Quantification of Angiogenic Factors

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Fifty micrograms of total cell lysate was separated on a 10% SDS-PAGE gel, transferred on PVDF membrane and blotted with antibodies. HGF, VEGFA, VEGFR2 and MMP2 antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). ERK1/2, phospho-ERK1/2 and α-Tubulin were from Cell Signaling and Sigma, respectively. HRP-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies were used to identify the protein of interest. Each experiment was repeated at least two times. Secreted VEGFA and HGF was detected either in conditioned media (CM) of cultured cells or patient's sera using ELISA kits from Ray Biotech (Norcross, GA, USA).

Ghosh A., Dasgupta D., Ghosh A., Roychoudhury S., Kumar D., Gorain M., Butti R., Datta S., Agarwal S., Gupta S., Krishna Dhali G., Chowdhury A., Schmittgen T.D., Kundu G.C, & Banerjee S. (2017). MiRNA199a-3p suppresses tumor growth, migration, invasion and angiogenesis in hepatocellular carcinoma by targeting VEGFA, VEGFR1, VEGFR2, HGF and MMP2. Cell Death & Disease, 8(3), e2706-.

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Liver Extracellular Matrix Characterization

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Liver materials used for light microscopy were stained with H&E for general histological evaluation. Electron micrographs of sample cross-sections were obtained at 5.0 kV and 500× magnification using a Hitachi S-4800 scanning electron microscope (Tokyo, Japan). GAGs were quantified using the Blyscan GAG assay kit (Biocolor, Belfast, UK) following the manufacturer’s instructions. The collagen content was quantified using a colorimetric assay to detect hydroxyproline following a modification of Grant’s method45 (link). Immunohistochemistry was performed to determine the retention of the important basement membrane proteins as well as important growth factors after the crosslinking process. The primary antibodies against collagen-I (1:1,000; GeneTex, Irvine, CA), fibronectin (1:50; Santa Cruz Biotechnology, Santa Cruz, CA), HGF (1:30; Santa Cruz Biotechnology, Santa Cruz, CA), VEGF (1:50; Santa Cruz Biotechnology, Santa Cruz, CA), bFGF (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) and IGF (1:50; Santa Cruz Biotechnology, Santa Cruz, CA) were used. Quantification was performed in at least 10 random views on each slide using Image Pro-plus software (5.0 ver., Media Cybernetics, Silver Spring, MD).

Wang Y., Bao J., Wu X., Wu Q., Li Y., Zhou Y., Li L, & Bu H. (2016). Genipin crosslinking reduced the immunogenicity of xenogeneic decellularized porcine whole-liver matrices through regulation of immune cell proliferation and polarization. Scientific Reports, 6, 24779.

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Renal Expression of Angiogenic Factors

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Standard blotting protocols were followed, as described^{9 (link), 18 (link), 43}, to determine renal expression of pro-angiogenic hypoxia-induced factor (HIF)-1α, VEGF (Santa Cruz, CA, 1:200), hepatocyte growth factor (HGF; Abcam, Cambridge, UK, 1:200), the specific VEGF and HGF receptors Flk-1 (Santa Cruz, CA, 1:200 for all) and cMet (Abbiotec, San Diego, CA, 1:200), total and phosphorylated (p)-eNOS (Cell Signaling, 1:500, Danvers, MA, 1:500), ERK ½ and akt (Santa Cruz, CA, 1:200).

Chade A.R., Williams M.L., Guise E., Vincent L.J., Harvey T.W., Kuna M., Mahdi F, & Bidwell GL I.I.I. (2017). Systemic biopolymer-delivered vascular endothelial growth factor promotes therapeutic angiogenesis in experimental renovascular disease. Kidney international, 93(4), 842-854.

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Muscle Tissue Protein Expression Analysis

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The lysates of muscle tissue samples on 1 w post injury were loaded on SDS-PAGE gel and transferred to the PVDF membrane. Then CD68 (1 : 1000, Abcam), iNOS (1 : 1000, Abcam), ArgI (1 : 1000, Abcam), TNF-α (1 : 1000, Abcam), α-SMA (1 : 1000, Cell Signaling Technology), desmin (1 : 50,000, Abcam), fibromodulin (FMOD, 1 : 1000, Abcam), myogenin (1 : 1000, Abcam), MyoD (1 : 100, Santa Cruz Biotech), collagen I (1 : 1000, BOSTER), HGF (1 : 100, Santa Cruz Biotech), AKT (1 : 1000, Cell Signaling Technology), pAKT (1 : 1000, Cell Signaling Technology), and GAPDH (1 : 20,000, Santa Cruz Biotech) were probed with the membrane as the primary antibodies at 4°C overnight. The membranes were rinsed and incubated with anti-mouse or anti-rabbit second antibodies at room temperature for 50 min. The blots were visualized by exposure to chemiluminescence ECL reagents (Beyotime). The gray values of the target bands were calculated by Image J software. Each result was obtained by at least three independent experiments.

Huang H., Liu J., Hao H., Chen D., Zhizhong L., Li M., Song H., Xiang R., Jiang C., Fu X, & Han W. (2017). Preferred M2 Polarization by ASC-Based Hydrogel Accelerated Angiogenesis and Myogenesis in Volumetric Muscle Loss Rats. Stem Cells International, 2017, 2896874.

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Immunofluorescence Staining of Liver Sections

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After the deparaffination of liver sections, heat‐induced target antigen retrieval was performed and the sections were blocked with protein block serum‐free blocking solution (DAKO, Glostrup, Denmark). Then, antibodies against active caspase‐3 (BD Biosciences), mouse α‐smooth muscle actin, and mouse F4/80 (Abcam Inc.), and mouse hepatocyte growth factor (HGF, Santa Cruz Biotechnology) were treated to the sections and incubated with Alexa 488‐conjugated donkey anti‐goat IgG, Alexa 555‐conjugated donkey anti‐rabbit IgG or Alexa 647‐conjugated donkey anti‐mouse IgG secondary antibody (Molecular Probes, Eugene, OR, USA). The nuclei were counter‐stained with Hoechst 33258 (Sigma) in the sections. All images were acquired using a Carl Zeiss LSM 700 light microscope (Carl Zeiss, Oberkochen, Germany).

Lee J., Kim S.R., Lee C., Jun Y.I., Bae S., Yoon Y.J., Kim O.Y, & Gho Y.S. (2021). Extracellular vesicles from in vivo liver tissue accelerate recovery of liver necrosis induced by carbon tetrachloride. Journal of Extracellular Vesicles, 10(10), e12133.

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Western Blot Analysis of Angiogenic Factors

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Samples were solubilized in lysis buffer [20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM phenylmethylsulfonyl fluoride, 1μg/ml leupeptin, and 2 μg/ml aprotinin] for 1 h at 4°C. The lysates were then clarified by centrifugation at 15,000 g for 30 min at 4°C, were diluted in Laemmli sample buffer containing 2% SDS and 5% (v/v) 2-mercaptoethanol, and were heated for 5 min at 90°C. The proteins were separated via SDS polyacrylamide gel electrophoresis (PAGE) using 10% or 15% resolving gels followed by transfer to nitrocellulose membranes (Bio-Rad, Hercules, CA) and then probed with antibodies against HIF-1a (Novus), CD31 (Abcam), HGF (Santa cruz), VEGF (Abcam), and FGF2 (Abcam) for 1h at room temperature (Table 2). Peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG and enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) were used as described by the manufacturer for detection. The membranes were scanned to create chemiluminescent images that were then quantified with an image analyzer (Kodak).

Park I.S., Chung P.S, & Ahn J.C. (2015). Enhancement of Ischemic Wound Healing by Spheroid Grafting of Human Adipose-Derived Stem Cells Treated with Low-Level Light Irradiation. PLoS ONE, 10(6), e0122776.

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Characterization of hAFSC and hAFSC-EV

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Whole cell lysates from hAFSC and hAFSC-EV were processed as previously described [39 (link)]. Primary antibodies were raised against the following molecules: HIF-1α, GAPDH, actin, HGF, IDO, lamin A/C (Santa Cruz Biotechnology, CA, USA), phospho-c-Kit^Tyr719 (Cell Signaling Technology, MA, USA), TGFβ (Novus, CO, USA), Rab5 (Lonza, SC, USA), CD9, and CD81 (Life Technologies, CA, USA).
Secondary antibodies, used at 1:3000 dilution, were all from Thermo Fisher Scientific (Waltham, MA, USA).

Casciaro F., Beretti F., Gatti M., Persico G., Bertucci E., Giorgio M, & Maraldi T. (2023). Effect of the Enrichment in c-Kit Stem Cell Potential of Foetal Human Amniotic Fluid Cells: Characterization from Single Cell Analysis to the Secretome Content. Biomedicines, 11(2), 430.

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Protein Expression Analysis in Angiogenic Signaling

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Equal amounts of protein (40 µg per lane) were electrophoresed on sodium dodecylsulfate-polyacrylamide gels (8% to 15%), transferred to polyvinylidene difluoride membrane, and probed with antibodies to HGF (Santa Cruz Biotechnology, Delaware CA, USA; 1:1000), Ang1 (Novus Biologicals), Ang2 (Novus Biologicals; 1:1000), phospho-eNOS (Signaling, Beverly, MA, USA; 1:250), eNOS (Cell Signaling; 1:500), phospho-Akt (Cell Signaling; 1:1000), Akt (Cell Signaling; 1:1000), phospho-ERK (Cell Signaling; 1:1000), ERK (Cell Signaling; 1:1000), NGF (Santa Cruz Biotechnology; 1:1000), NT-3 (Santa Cruz Biotechnology; 1:1000), GAPDH (ABclonal, Woburn, MA, USA; 1:5000), or β-actin (Abcam; 1:6000). The results were quantified by densitometry (N = 4 per group).

Kwon M.H., Song K.M., Limanjaya A., Choi M.J., Ghatak K., Nguyen N.M., Ock J., Yin G.N., Kang J.H., Lee M.R., Gho Y.S., Ryu J.K, & Suh J.K. (2019). Embryonic stem cell-derived extracellular vesicle-mimetic nanovesicles rescue erectile function by enhancing penile neurovascular regeneration in the streptozotocin-induced diabetic mouse. Scientific Reports, 9, 20072.

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Protein Expression Analysis of Myogenic Differentiation

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Immunoblots were carried out using protein lysates obtained from undifferentiated pPICs and pPICs that had undergone early myogenic differentiation (n = 3/group). Approximately, 50 μg of protein were separated on gradient (10% to 15%) SDS-polyacrylamide gels. After electrophoresis, proteins were transferred onto nitrocellulose membranes and blocked with 5% milk, then incubated with antibodies against IGF-1 (Santa Cruz Biotechnology, Dallas, Texas), HGF (Santa Cruz Biotechnology), transforming growth factor (TGF)-β1 (Santa Cruz Biotechnology), neuregulin (NRG)-1 (Santa Cruz Biotechnology) at dilutions suggested by the manufacturers. GAPDH (Millipore) was used as a loading control. Proteins were detected by chemiluminescence using horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology) and visualized using ECL Plus Western Blotting Detection Reagents (Amersham, Little Chalfont, United Kingdom) and a Chemidoc imaging system (Bio-Rad).

Lewis F.C., Cottle B.J., Shone V., Marazzi G., Sassoon D., Tseng C.C., Dankers P.Y., Chamuleau S.A., Nadal-Ginard B, & Ellison-Hughes G.M. (2017). Transplantation of Allogeneic PW1pos/Pax7neg Interstitial Cells Enhance Endogenous Repair of Injured Porcine Skeletal Muscle. JACC: Basic to Translational Science, 2(6), 717-736.

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Protein Profiling of Extracellular Vesicles, Cells, and Tissues

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Total proteins from EVs, cASCs, and colon tissues were extracted using the PRO-PREP Protein Extraction Kit (iNtRON Biotechnology, Seongnam, South Korea) and measured using the Bio-Rad DC Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). The total protein content in each 20 μg sample was subjected to SDS-PAGE and immunoblotting with antibodies against TSG-6 (Santa Cruz Biotechnology, CA, USA), COX-2(Santa Cruz Biotechnology), HGF, beta-actin(Santa Cruz Biotechnology), CD206 (Santa Cruz Biotechnology), Arginase (Santa Cruz Biotechnology), FOXP3 (Santa Cruz Biotechnology), TGF-β (Cusabio Biotech, Wuhan, China), CD63 (LSBio, Seattle, WA, USA), CD9 (GeneTex, Irvine, CA, USA), beta-actin (Santa Cruz Biotechnology), TNF-α (Cusabio Biotech), IL-6 (Cusabio Biotech), IL-10 (ABclonal Tech, MA, USA) and IL-17 (ABclonal Tech).

An J.H., Li Q., Bhang D.H., Song W.J, & Youn H.Y. (2020). TNF-α and INF-γ primed canine stem cell-derived extracellular vesicles alleviate experimental murine colitis. Scientific Reports, 10, 2115.

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