Human Pref-1/DLK1/FA1 Antibody

Detection of Human Pref‑1/DLK1/FA1 by Western Blot. Western blot shows lysates of human adrenal gland tissue and human placenta tissue. PVDF membrane was probed with 1 µg/mL of Goat Anti-Human Pref-1/DLK1/FA1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1144) followed by HRP-conjugated Anti-Goat IgG Secondary Antibody (Catalog # HAF017). A specific band was detected for Pref-1/DLK1/FA1 at approximately 45-60 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 1.

Pref‑1/DLK1/FA1 in HepG2 Human Cell Line. Pref-1/DLK1/FA1 was detected in immersion fixed HepG2 human hepatocellular carcinoma cell line using Goat Anti-Human Pref-1/DLK1/FA1 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF1144) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog # NL001) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm. View our protocol for Fluorescent ICC Staining of Cells on Coverslips.

Detection of Mouse Pref-1/DLK1/FA1 by Western Blot Dlk1 inhibits myoblast differentiation in vitro.A: C2C12 cells were constructed to constitutively express full-length Dlk1 under control by the CMV promotor, DLK1-C2C12. Cell morphology and myogenic differentiation potential was analyzed by immunocytochemistry. Forced expression of Dlk1 resulted in inhibition of myogenic differentiation observed as no formation of myotubes, lack of myostatin expression and reduced Desmin staining compared to C2C12 parental control cells. Addition of Dlk1 antibody to the culture supernatant during differentiation reverted the effect of forced Dlk1 expression observed as a regained ability to form myotubes and express myogenin compared to control cells. Scalebars: 20 µm in all images. B: DLK1-C2C12 cells (DLK1) and C2C12 parental control cells were cultured under proliferating conditions for 3 days followed by differentiation for another 5 days. Cells were harvested and analyzed at 1, 3, 5, and 8 days in culture. The differentiation process was analyzed by qPCR of Dlk1, the myogenic markers Mef2a, Myf5, Myod, and Myogenin in addition to Myostatin, Follistatin, TGF beta 1, Smad7 and Decorin. All qPCR analyses were run in triplicates on duplicate analyses containing triplicate samples (n = 2 for each time point) and presented as relative expression levels with normalization to.Actb, Gusb, Pgk1, Gapdh and Tfrc as described in materials and methods. Black bars: DLK1+ cells; white bars: control cells. C: Dlk1 (13 kDa band) and myostatin (a double band corresponding to 40–50 kDa) protein level was analyzed with western blotting during proliferation and differentiation of DLK1-C2C12 and control cells. Dlk1 protein was only expressed by DLK1-C2C12 cells and not by control cells. Myostatin protein was expressed in control cells during differentiation but not during proliferation of either of the cells lines or during differentiation of DLK1-C2C12 cells. However, addition of Dlk1 antibody resulted in expression of myostatin by the DLK1-C2C12 cells during differentiation. Beta-actin (45 kDa) was used as control. Image collected and cropped by CiteAb from the following open publication (https://dx.plos.org/10.1371/journal.pone.0060692), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Pref-1/DLK1/FA1 by Immunocytochemistry/ Immunofluorescence Dlk1 inhibits myoblast differentiation in vitro.A: C2C12 cells were constructed to constitutively express full-length Dlk1 under control by the CMV promotor, DLK1-C2C12. Cell morphology and myogenic differentiation potential was analyzed by immunocytochemistry. Forced expression of Dlk1 resulted in inhibition of myogenic differentiation observed as no formation of myotubes, lack of myostatin expression and reduced Desmin staining compared to C2C12 parental control cells. Addition of Dlk1 antibody to the culture supernatant during differentiation reverted the effect of forced Dlk1 expression observed as a regained ability to form myotubes and express myogenin compared to control cells. Scalebars: 20 µm in all images. B: DLK1-C2C12 cells (DLK1) and C2C12 parental control cells were cultured under proliferating conditions for 3 days followed by differentiation for another 5 days. Cells were harvested and analyzed at 1, 3, 5, and 8 days in culture. The differentiation process was analyzed by qPCR of Dlk1, the myogenic markers Mef2a, Myf5, Myod, and Myogenin in addition to Myostatin, Follistatin, TGF beta 1, Smad7 and Decorin. All qPCR analyses were run in triplicates on duplicate analyses containing triplicate samples (n = 2 for each time point) and presented as relative expression levels with normalization to.Actb, Gusb, Pgk1, Gapdh and Tfrc as described in materials and methods. Black bars: DLK1+ cells; white bars: control cells. C: Dlk1 (13 kDa band) and myostatin (a double band corresponding to 40–50 kDa) protein level was analyzed with western blotting during proliferation and differentiation of DLK1-C2C12 and control cells. Dlk1 protein was only expressed by DLK1-C2C12 cells and not by control cells. Myostatin protein was expressed in control cells during differentiation but not during proliferation of either of the cells lines or during differentiation of DLK1-C2C12 cells. However, addition of Dlk1 antibody resulted in expression of myostatin by the DLK1-C2C12 cells during differentiation. Beta-actin (45 kDa) was used as control. Image collected and cropped by CiteAb from the following open publication (https://dx.plos.org/10.1371/journal.pone.0060692), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Mouse Pref-1/DLK1/FA1 by Immunocytochemistry/ Immunofluorescence Dlk1 inhibits myoblast differentiation in vitro.A: C2C12 cells were constructed to constitutively express full-length Dlk1 under control by the CMV promotor, DLK1-C2C12. Cell morphology and myogenic differentiation potential was analyzed by immunocytochemistry. Forced expression of Dlk1 resulted in inhibition of myogenic differentiation observed as no formation of myotubes, lack of myostatin expression and reduced Desmin staining compared to C2C12 parental control cells. Addition of Dlk1 antibody to the culture supernatant during differentiation reverted the effect of forced Dlk1 expression observed as a regained ability to form myotubes and express myogenin compared to control cells. Scalebars: 20 µm in all images. B: DLK1-C2C12 cells (DLK1) and C2C12 parental control cells were cultured under proliferating conditions for 3 days followed by differentiation for another 5 days. Cells were harvested and analyzed at 1, 3, 5, and 8 days in culture. The differentiation process was analyzed by qPCR of Dlk1, the myogenic markers Mef2a, Myf5, Myod, and Myogenin in addition to Myostatin, Follistatin, TGF beta 1, Smad7 and Decorin. All qPCR analyses were run in triplicates on duplicate analyses containing triplicate samples (n = 2 for each time point) and presented as relative expression levels with normalization to.Actb, Gusb, Pgk1, Gapdh and Tfrc as described in materials and methods. Black bars: DLK1+ cells; white bars: control cells. C: Dlk1 (13 kDa band) and myostatin (a double band corresponding to 40–50 kDa) protein level was analyzed with western blotting during proliferation and differentiation of DLK1-C2C12 and control cells. Dlk1 protein was only expressed by DLK1-C2C12 cells and not by control cells. Myostatin protein was expressed in control cells during differentiation but not during proliferation of either of the cells lines or during differentiation of DLK1-C2C12 cells. However, addition of Dlk1 antibody resulted in expression of myostatin by the DLK1-C2C12 cells during differentiation. Beta-actin (45 kDa) was used as control. Image collected and cropped by CiteAb from the following open publication (https://dx.plos.org/10.1371/journal.pone.0060692), licensed under a CC-BY license. Not internally tested by R&D Systems.