Journal: The Journal of Biological Chemistry

Article Title: Matriptase-2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway

doi: 10.1074/jbc.M117.801795

Figure Lengend Snippet: Mouse ORF constructs used for transfection studies

Article Snippet: cDNA constructs We purchased mouse MT2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_027902.1","term_id":"27229128","term_text":"NM_027902.1"}} NM_027902.1 ), ALK2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_007394.1","term_id":"6680627","term_text":"NM_007394.1"}} NM_007394.1 ), ALK3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_009758.3","term_id":"46519167","term_text":"NM_009758.3"}} NM_009758.3 ), ActRIIA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_007396.1","term_id":"6680631","term_text":"NM_007396.1"}} NM_007396.1 ), Bmpr2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_007561.3","term_id":"145966831","term_text":"NM_007561.3"}} NM_007561.3 ), and Hfe ( {"type":"entrez-nucleotide","attrs":{"text":"NM_010424.1","term_id":"6754189","term_text":"NM_010424.1"}} NM_010424.1 ) ORF with a C-terminal FLAG/MYC epitope in pCMV6 vector (pCMV6-MT2, ALK2, ALK3, ActRIIA, Bmpr2, and Hfe) from Origene ( ).

Techniques: Construct, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Matriptase-2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway

doi: 10.1074/jbc.M117.801795

Figure Lengend Snippet: MT2 cleaves ALK2 and ALK3 and decreases their full-length forms on cell surface. A, a representative diagram for all BMP receptors used in this study, including ALK2, ALK3, ActRIIA, and Bmpr2. All these receptors are type I transmembrane proteins with a short extracellular domain (∼120–150 amino acids), a transmembrane domain (PM), a large intracellular domain, and a C-terminal FLAG/Myc epitope (f/m). B, MT2 cleaves ALK2 in HEK293 cells. HEK293 cells in 12-well plates were co-transfected with an equal amount of pCMV6-ALK2 (2 μg), increasing amounts of pCMV6-MT2 or S762A-MT2 (0, 0.25, 0.5, 1.0, and 2.0 μg), and decreasing amounts of pEGFP-N1 (2.0, 1.75, 1.5, 1.0, and 0 μg). After 48 h of transfection, MT2 and ALK2 in ∼150 μg of cell lysate proteins were immunodetected by using anti-FLAG antibody, and β-actin and EGFP by specific antibodies. HEK293 cells (Ctrl) were included as a negative control. C, MT2 decreases full-length ALK2 on the cell surface. Co-transfection of HEK293 cells with pCMV6-ALK2 and pEGFP-N1, pCMV6-MT2, or S762A-MT2, and biotinylation of cell-surface proteins were performed essentially the same as described in the legend to Fig. 5C. The eluted cell-surface proteins and input lysate were subjected to SDS-PAGE and immunodetection of ALK2 and MT2 using an anti-FLAG antibody, and Na+K+-ATPase (NaK ATPase), β-actin, and EGFP by using specific antibodies. D and E, MT2 cleaves ALK3 in HEK293 cells (D) and decreases the full-length ALK3 on the cell surface (E). The experiments were performed as described above for ALK2 in B and C. ALK3 was immunodetected by using an anti-FLAG antibody. Two MT2/ALK3 images with different exposure times were presented (D). All experiments were repeated at least three times (technical replicate = 1; independent biological replicates ≥ 3) with consistent results.

Article Snippet: cDNA constructs We purchased mouse MT2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_027902.1","term_id":"27229128","term_text":"NM_027902.1"}} NM_027902.1 ), ALK2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_007394.1","term_id":"6680627","term_text":"NM_007394.1"}} NM_007394.1 ), ALK3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_009758.3","term_id":"46519167","term_text":"NM_009758.3"}} NM_009758.3 ), ActRIIA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_007396.1","term_id":"6680631","term_text":"NM_007396.1"}} NM_007396.1 ), Bmpr2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_007561.3","term_id":"145966831","term_text":"NM_007561.3"}} NM_007561.3 ), and Hfe ( {"type":"entrez-nucleotide","attrs":{"text":"NM_010424.1","term_id":"6754189","term_text":"NM_010424.1"}} NM_010424.1 ) ORF with a C-terminal FLAG/MYC epitope in pCMV6 vector (pCMV6-MT2, ALK2, ALK3, ActRIIA, Bmpr2, and Hfe) from Origene ( ).

Techniques: Transfection, Negative Control, Cotransfection, SDS Page, Immunodetection

Journal: The Journal of Biological Chemistry

Article Title: Matriptase-2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway

doi: 10.1074/jbc.M117.801795

Figure Lengend Snippet: MT2 cleaves AcRIIA and Bmpr2 and decreases their full-length forms on cell surface. A and B, MT2 cleaves ActRIIA (A) and decreases the full-length ActRIIA on the cell surface (B). Co-transfection of HEK293 cells with pCMV6-ActRIIA and pEGFP-N1, pCMV6-MT2, or S762A-MT2, biotinylation of cell-surface proteins, and immunodetection were performed essentially the same as described in the legend to Fig. 6, B and C. ActRIIA was detected by using an anti-FLAG antibody. C and D, MT2 cleaves Bmpr2 (C) and decreases the full-length Bmpr2 on cell surface (D). Experiments were performed as described above for ActRIIA. Bmpr2 was detected by using an anti-FLAG antibody. Two MT2/Bmpr2 images with different exposure times were presented (C). E and F, MT2 does not cleave Zip14 (E) and does not decrease cell-surface Zip14 (F). The experiments were performed as described above for ActRIIA and Bmpr2. Zip14 was immunodetected by using an anti-FLAG antibody. All experiments were repeated at least three times (technical replicate = 1; independent biological replicates ≥3) with consistent results.

Article Snippet: cDNA constructs We purchased mouse MT2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_027902.1","term_id":"27229128","term_text":"NM_027902.1"}} NM_027902.1 ), ALK2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_007394.1","term_id":"6680627","term_text":"NM_007394.1"}} NM_007394.1 ), ALK3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_009758.3","term_id":"46519167","term_text":"NM_009758.3"}} NM_009758.3 ), ActRIIA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_007396.1","term_id":"6680631","term_text":"NM_007396.1"}} NM_007396.1 ), Bmpr2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_007561.3","term_id":"145966831","term_text":"NM_007561.3"}} NM_007561.3 ), and Hfe ( {"type":"entrez-nucleotide","attrs":{"text":"NM_010424.1","term_id":"6754189","term_text":"NM_010424.1"}} NM_010424.1 ) ORF with a C-terminal FLAG/MYC epitope in pCMV6 vector (pCMV6-MT2, ALK2, ALK3, ActRIIA, Bmpr2, and Hfe) from Origene ( ).

Techniques: Cotransfection, Immunodetection

Journal: The Journal of Biological Chemistry

Article Title: Matriptase-2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway

doi: 10.1074/jbc.M117.801795

Figure Lengend Snippet: Holo-Tf blocks MT2 cleavage of Tfr2 (A), but not ALK2 (B), ALK3 (C), ActRIIA (D), Bmpr2 (E), or Hfe (F). pcDNA3-Tfr2, pCMV6-ALK2, ALK3, ActRIIA, Bmpr2, or Hfe/B2M were co-transfected into HEK293 cells with an equal amount of pEGFP-N1 or pCMV6-MT2. At about 24 h post-transfection, medium was changed to Opti-MEM, 1% FCS with or without 30 μm holo-Tf (Tf). After another 24 h of incubation, cell lysate was collected for immunodetection of MT2, ALK2, ALK3, ActRIIA, Bmpr2, and Hfe by using an anti-FLAG antibody, and Tfr2, β-actin, and ferritin by using specific antibodies. All experiments were repeated at least three times (technical replicate = 1; independent biological replicates ≥3) with consistent results. G, a model for MT2 suppression of hepcidin expression. Bmp receptors, Hjv, Hfe, and Tfr2 form a complex with BMP6 at the plasma membrane to induce hepcidin expression through phosphorylation of SMAD1, -5, and -8. MT2 suppresses hepcidin expression by cleaving the extracellular portion of these membrane proteins.

Article Snippet: cDNA constructs We purchased mouse MT2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_027902.1","term_id":"27229128","term_text":"NM_027902.1"}} NM_027902.1 ), ALK2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_007394.1","term_id":"6680627","term_text":"NM_007394.1"}} NM_007394.1 ), ALK3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_009758.3","term_id":"46519167","term_text":"NM_009758.3"}} NM_009758.3 ), ActRIIA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_007396.1","term_id":"6680631","term_text":"NM_007396.1"}} NM_007396.1 ), Bmpr2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_007561.3","term_id":"145966831","term_text":"NM_007561.3"}} NM_007561.3 ), and Hfe ( {"type":"entrez-nucleotide","attrs":{"text":"NM_010424.1","term_id":"6754189","term_text":"NM_010424.1"}} NM_010424.1 ) ORF with a C-terminal FLAG/MYC epitope in pCMV6 vector (pCMV6-MT2, ALK2, ALK3, ActRIIA, Bmpr2, and Hfe) from Origene ( ).

Techniques: Transfection, Incubation, Immunodetection, Expressing

Journal: Neural Development

Article Title: Inductive specification and axonal orientation of spinal neurons mediated by divergent bone morphogenetic protein signaling pathways

doi: 10.1186/1749-8104-6-36

Figure Lengend Snippet: Type II BMP receptor expression in dI neurons . (A) Western analysis of whole cell lysates of dissociated dI neurons probed with α-BMPRII (130 kDa, lane 1), α-ActRIIA/B (70 kDa, lane 2) and α-ActRIIB (58 kDa, lane 3) antibodies. (B) Immunofluorescence labeling of dissociated dI neurons with antibodies against type II BMP receptors (α-BMPRII, α-ActRIIA/B and α-ActRIIB). The left column shows relative numbers of labeled neurons against background low level fluorescence (scale = 50 μm). The center column (scale = 20 μm) and right column (scale = 10 μm) highlight neuronal detail. (C) Direct comparison of ActRIIA and ActRIIB expression in sister cultures. Phase contrast (left), BMP receptor immunofluorescence labeling (center) and DAPI nuclear staining (right) of dissociated dI neurons. Arrowheads indicate ActRIIB + growth cones. Analysis of immunofluorescence label for each antibody from three to four 20 × fields showed that 85.6 ± 3.7% of 1,056 total neurons were ActRIIA + (n = 4), 33.1 ± 3.7% of 732 total neurons were ActRIIB + (n = 4) and 78.6 ± 3.3% of 465 total neurons were BMPRII + (n = 3). Scale = 50 μm.

Article Snippet: Antibodies were: mouse α-TAG-1 (4D7 [ ]); rabbit α-Lhx2/9 (L1 [ ]); mouse α-ERM (13H9 [ ]); rabbit α-Smad1/5/8 (N18; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit α-phospho-Smad1/5/8 (pSmad); rabbit α-phospho-Akt(S) (pAkt) and rabbit α-Akt (Cell Signaling Technology); mouse α-flag (M2; Sigma); rat α-Netrin-1 (R&D Systems); rabbit α-ActRII (H65) and goat α-ActRIIB (N16) (Santa Cruz); mouse α-ActRIIB (abcam, Cambridge, MA USA); and mouse α-BMPRII (BD Transduction Laboratories, San Jose, CA, USA).

Techniques: Expressing, Western Blot, Immunofluorescence, Labeling, Fluorescence, Staining