alk6  (R&D Systems)

Journal: Biology

Article Title: BMP-2-Driven Osteogenesis: A Comparative Analysis of Porcine BMSCs and ASCs and the Role of TGF-β and FGF Signaling.

doi: 10.3390/biology14060610

Figure Lengend Snippet: Figure 6. Receptor expressions of ALK2, ALK4, ALK6, ALK5, ALK7, BMPR-II in pASC. Grouped representation of the respective receptor expression in the course of osteogenic differentiation (OM +/−BMP-2). From day 19, there is a significant induction of ALK 2, ALK 6, and ALK 5 with the addition of BMP-2. BMPR-II expression in the OM group decreased in OM and tended to stay increased under BMP-2 supplementation from day 19, but was not considered significant (* p ≤0.05, ** p ≤0.01; n = 6, BMP-2 450 ng/mL).

Article Snippet: The respective conjugated antibodies were used for the expressions of ALK3 (Cat. No.: AF436), ALK 5 (Cat. No.: FAB5871), ALK6 (Cat. No.: FAB5051A), TGF-β2-RII (Cat. No.: FAB532P), ALK7 (Cat. No.: FAB77491A), ALK2 (Cat. No.: AF637), ALK4 (Cat. No.: MAB2221), and BMPR-II (Cat. No.: AF811) (by R&D Systems, Minneapolis, MN, USA), and the pASCs and pBMSCs were compared for their expressions of the specific surface antigens CD45 (Cat. No.: MCA1568GA, BioRad, Hercules, CA, USA), HLA-DR (human leukocyte antigen–antigen D-related surface molecule) (Cat. No.: MCA2314F, Bio-Rad, Hercules, CA, USA), CD29 (Cat. No.: 561,496, BD Pharmingen, Franklin Lakes, NJ, USA), CD79alpha (Bio-Rad, Cat. No.: MCA2538GA), CD14 (Cat. No.: MCA1568GA, Bio-Rad, Hercules, CA, USA), CD31 (Cat. No.: AF3387, R&D Systems, Minneapolis, MN, USA), CD105 (Cat. No.: NB110-58718APC, Novus Biologicals, Minneapolis, MN, USA), CD26 (, Cat. No.: NB600-552APC, Novus Biologicals, Minneapolis, MN, USA), CD73 (, Cat. No.: AF4488, R&D Systems, Minneapolis, MN, USA), CD90 (Cat. No.: 559,869, BD Pharmingen, Franklin Lakes, NJ, USA), CD34 (Cat. No.: 81289, abcam, Cambridge, UK), and CD44 (Cat. No.: 5531, BD Pharmingen, Franklin Lakes, NJ, USA).

Techniques: Expressing

Journal: Molecular cancer research : MCR

Article Title: BMP7 Signaling in TGFBR2 -deficient Stromal Cells Provokes Epithelial Carcinogenesis

doi: 10.1158/1541-7786.MCR-18-0120

Figure Lengend Snippet: (A) Western blots analyses for Smad2 and Smad1/5 phosphorylation levels in forestomach tissue lysate from approximately 6-week-old mice with the listed genotypes, 30 µg of protein loaded per lane, each lane represents a distinct mouse. (B) RT-PCR electrophoretic product for the listed genes in the forestomach of approximately 6-week-old mice with the listed genotypes. Buffer: no template control. (C) RT-PCR electrophoretic product for HGF in the forestomach fibroblasts from approximately 6-week-old mice with the listed genotypes with or without (0.1% FBS) stimulation with BMP7 (100 ng.ml−1). (D) Western blot analysis for BMP7 in forestomach tissue lysate from approximately 6-week-old mice with the listed genotypes, 30 µg of protein loaded per lane, each lane represents a distinct mouse. The actin blot is the same as depicted in Fig. 2G. (E) In situ hybridization for HGF and BMP7 mRNA (NBT/BCTP substrate, purple) in sections of the forestomach of the listed mice. L: lumen, S: stroma, E: epithelium/cancer cells, M: smooth muscle. Arrows point to stromal HGF and cancer cell BMP7 expression, respectively. Scale bars: upper panel (HGF): 50 µm, lower panel (BMP7): 20 µm. (F) Immunolabeling for BMP7, ALK6, ALK6 & FSP1. Color substrate: 3, 3´-diaminobenzidine (DAB, brown); for ALK6 & FSP1: 3-amino-9-ethylcarbazole (AEC, red) and Vector Blue. Scale bar: 20 µm. S: stroma. Arrowhead points to FSP1+ fibroblast with ALK6 immunoreactivity. (G) Immunofluorescence for ALK6 in cultured forestomach fibroblasts from approximately 6-week-old mice with the listed genotypes. Scale bars: 50 µm. Red inset: negative control, scale bar: 20 µm.

Article Snippet: Goat anti-BMP7 (Santa Cruz Biotechnology, sc-6899, dilution: 1:25), goat anti-ALK2 (R&D, AF637, 1:50), rabbit anti-ALK3 (Santa Cruz, sc-20736, 1:150), goat anti-ALK6 (Santa Cruz, sc-5679, 1:150), rat anti-CD45 (R&D, MAB114, 1:50), rabbit anti-CK5 (Abcam, ab24647, 1:1200), rabbit anti-CK20 (Abcam, ab53120, 1:200), rabbit anti-Met (phosphorylated Tyr1001, Abcam, ab61024, 1:50), rabbit anti-S100A4/FSP1 (gift from E.G. Neilson, 1:450), rabbit anti-HGF (Santa Cruz, sc-7949, 1:50), rabbit anti-Ki67 (Abcam, ab15580, 1:500) and rabbit anti-p63 (phosphorylated Ser160/162, Cell Signaling, #4981, 1:100).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Expressing, Immunolabeling, Plasmid Preparation, Immunofluorescence, Cell Culture, Negative Control

Journal: Molecular cancer research : MCR

Article Title: BMP7 Signaling in TGFBR2 -deficient Stromal Cells Provokes Epithelial Carcinogenesis

doi: 10.1158/1541-7786.MCR-18-0120

Figure Lengend Snippet: (A) Cell proliferation (methylene blue absorbance) of wt (gray), TGFBR2cKO (red) or TGFBR2/SMAD4cKO (blue) forestomach fibroblasts cultured in vitro with the indicated treatments. The data is normalized by setting the 0.1% FBS control at 1. Unpaired one-tailed t test compared to 0.1% FBS control, statistical analyses carried out on untransformed data. The data is presented as the mean ± SEM. *p<0.05, **p<0.01, ns: not significant. See accompanying source data. (B) Diagram of the proposed crosstalk between the TGFBR2-deficient FSP1+ fibroblasts (blue) and the overlying epithelium (black). FSP1+ fibroblasts produce HGF to activate Met receptors on overlying epithelial cells, causing epithelial cells to produce BMP7, which stimulates ALK6 receptors on FSP1+ fibroblasts to produce HGF. (C) Comparison of BMP7 and HGF protein levels in forestomach whole tissue lysates of wt, TGFβR2floxE2; αSMA-Cre and TGFβR2floxE2; FSP1-Cre (TGFBR2cKO) mice. Western blots, 30 µg of protein loaded per lane. BMP7 and HGF are highly upregulated in the TGFBR2cKO forestomach cancer tissue.

Article Snippet: Goat anti-BMP7 (Santa Cruz Biotechnology, sc-6899, dilution: 1:25), goat anti-ALK2 (R&D, AF637, 1:50), rabbit anti-ALK3 (Santa Cruz, sc-20736, 1:150), goat anti-ALK6 (Santa Cruz, sc-5679, 1:150), rat anti-CD45 (R&D, MAB114, 1:50), rabbit anti-CK5 (Abcam, ab24647, 1:1200), rabbit anti-CK20 (Abcam, ab53120, 1:200), rabbit anti-Met (phosphorylated Tyr1001, Abcam, ab61024, 1:50), rabbit anti-S100A4/FSP1 (gift from E.G. Neilson, 1:450), rabbit anti-HGF (Santa Cruz, sc-7949, 1:50), rabbit anti-Ki67 (Abcam, ab15580, 1:500) and rabbit anti-p63 (phosphorylated Ser160/162, Cell Signaling, #4981, 1:100).

Techniques: Cell Culture, In Vitro, One-tailed Test, Western Blot

Journal: Molecular cancer research : MCR

Article Title: BMP7 Signaling in TGFBR2 -deficient Stromal Cells Provokes Epithelial Carcinogenesis

doi: 10.1158/1541-7786.MCR-18-0120

Figure Lengend Snippet: (A) Western blots analyses for Smad2 and Smad1/5 phosphorylation levels in forestomach tissue lysate from approximately 6-week-old mice with the listed genotypes, 30 µg of protein loaded per lane, each lane represents a distinct mouse. (B) RT-PCR electrophoretic product for the listed genes in the forestomach of approximately 6-week-old mice with the listed genotypes. Buffer: no template control. (C) RT-PCR electrophoretic product for HGF in the forestomach fibroblasts from approximately 6-week-old mice with the listed genotypes with or without (0.1% FBS) stimulation with BMP7 (100 ng.ml−1). (D) Western blot analysis for BMP7 in forestomach tissue lysate from approximately 6-week-old mice with the listed genotypes, 30 µg of protein loaded per lane, each lane represents a distinct mouse. The actin blot is the same as depicted in Fig. 2G. (E) In situ hybridization for HGF and BMP7 mRNA (NBT/BCTP substrate, purple) in sections of the forestomach of the listed mice. L: lumen, S: stroma, E: epithelium/cancer cells, M: smooth muscle. Arrows point to stromal HGF and cancer cell BMP7 expression, respectively. Scale bars: upper panel (HGF): 50 µm, lower panel (BMP7): 20 µm. (F) Immunolabeling for BMP7, ALK6, ALK6 & FSP1. Color substrate: 3, 3´-diaminobenzidine (DAB, brown); for ALK6 & FSP1: 3-amino-9-ethylcarbazole (AEC, red) and Vector Blue. Scale bar: 20 µm. S: stroma. Arrowhead points to FSP1+ fibroblast with ALK6 immunoreactivity. (G) Immunofluorescence for ALK6 in cultured forestomach fibroblasts from approximately 6-week-old mice with the listed genotypes. Scale bars: 50 µm. Red inset: negative control, scale bar: 20 µm.

Article Snippet: For immunofluorescence Anti-ALK6 (Santa Cruz, sc-25455, 1:50), anti-CK5 (Abcam, ab24647, 1:300), anti-Met (phosphorylated Tyr1001, Abcam, ab61024, 1:50), anti-FSP1 (gift from E.G. Neilson, 1:150), anti-HGFα (Santa Cruz, sc-7949, 1:50), anti-Tgfbr2 (Santa Cruz, sc-220, 1:100), anti-S100A4/FSP1 (DAKO, A5114, 1:4000) or anti-Ki67 (Thermo Scientific, RM-9106-S, 1:500).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, In Situ Hybridization, Expressing, Immunolabeling, Plasmid Preparation, Immunofluorescence, Cell Culture, Negative Control

Journal: Molecular cancer research : MCR

Article Title: BMP7 Signaling in TGFBR2 -deficient Stromal Cells Provokes Epithelial Carcinogenesis

doi: 10.1158/1541-7786.MCR-18-0120

Figure Lengend Snippet: (A) Cell proliferation (methylene blue absorbance) of wt (gray), TGFBR2cKO (red) or TGFBR2/SMAD4cKO (blue) forestomach fibroblasts cultured in vitro with the indicated treatments. The data is normalized by setting the 0.1% FBS control at 1. Unpaired one-tailed t test compared to 0.1% FBS control, statistical analyses carried out on untransformed data. The data is presented as the mean ± SEM. *p<0.05, **p<0.01, ns: not significant. See accompanying source data. (B) Diagram of the proposed crosstalk between the TGFBR2-deficient FSP1+ fibroblasts (blue) and the overlying epithelium (black). FSP1+ fibroblasts produce HGF to activate Met receptors on overlying epithelial cells, causing epithelial cells to produce BMP7, which stimulates ALK6 receptors on FSP1+ fibroblasts to produce HGF. (C) Comparison of BMP7 and HGF protein levels in forestomach whole tissue lysates of wt, TGFβR2floxE2; αSMA-Cre and TGFβR2floxE2; FSP1-Cre (TGFBR2cKO) mice. Western blots, 30 µg of protein loaded per lane. BMP7 and HGF are highly upregulated in the TGFBR2cKO forestomach cancer tissue.

Article Snippet: For immunofluorescence Anti-ALK6 (Santa Cruz, sc-25455, 1:50), anti-CK5 (Abcam, ab24647, 1:300), anti-Met (phosphorylated Tyr1001, Abcam, ab61024, 1:50), anti-FSP1 (gift from E.G. Neilson, 1:150), anti-HGFα (Santa Cruz, sc-7949, 1:50), anti-Tgfbr2 (Santa Cruz, sc-220, 1:100), anti-S100A4/FSP1 (DAKO, A5114, 1:4000) or anti-Ki67 (Thermo Scientific, RM-9106-S, 1:500).

Techniques: Cell Culture, In Vitro, One-tailed Test, Western Blot