Journal: Cell reports

Article Title: Netrin1 patterns the dorsal spinal cord through modulation of Bmp signaling

doi: 10.1016/j.celrep.2024.114954

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti Phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser465/467) (D5B10) , Cell Signaling Technology , Cat#11971; RRID:AB_2797785.

Techniques: Recombinant, Protease Inhibitor, Labeling, Gene Expression, In Situ, Sequencing, Software, Western Blot, Imaging

Journal: Cell reports

Article Title: Netrin1 patterns the dorsal spinal cord through modulation of Bmp signaling

doi: 10.1016/j.celrep.2024.114954

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti Phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465)/Smad8 (Ser465/467) (D5B10) , Cell Signaling Technology , Cat#11971; RRID:AB_2797785.

Techniques: Recombinant, Protease Inhibitor, Labeling, Gene Expression, In Situ, Sequencing, Software, Western Blot, Imaging

Journal: Theranostics

Article Title: Interleukin-34 orchestrates bone formation through its binding to bone morphogenic proteins

doi: 10.7150/thno.107340

Figure Lengend Snippet: The interaction IL34-BMP2 modulates SMAD1/5 as well as MCSF receptor (MCSFR) phosphorylation and related signaling. (A) Western blot analysis of SMDA1/5 and SMAD2 phosphorylations of human MNNG-HOS osteosarcoma cells in the presence of BMP2 (10 ng/mL), TGFß (10 ng/mL), MCSF (20 ng/mL), IL34 (20 ng/mL) alone or in corresponding combination. A representative experiment is shown. CT: basic culture medium. (B) Western blot analysis of SMDA1/5 phosphorylation of human MNNG-HOS osteosarcoma cells in the presence of BMP2 (10 ng/mL), IL34 (20 ng/mL) alone or in combination (BMP2+IL34, -) plus the human IL34 blocking antibody (BT34) (100 µg/mL) or its irrelevant isotypic control antibody (ISO) (100 µg/mL). CT: basic culture medium. (C) Western blot analysis of SMDA1/5, similar conditions used in B in the presence of the human IL34 blocking antibody (BT34) (100 µg/mL) or the natural inhibitor of BMP2 called NOGGIN (NOG) (200 ng/mL). (D) Kinetic analysis by Western blot of the potentiating effect of IL34 on BMP2-induced SMAD1/5 phosphorylation at 15 min, 30 min and 60 min with similar corresponding molecules concentrations described in B. (E) Western blot analysis of SMDA1/5 as described in B in the presence of a single concentration of BMP2 (10 ng/mL) in combination with gradual quantities of IL34 (10, 20, 40, 80 and 100 ng/mL). (F) The Alpha SureFire technology (Revvity) was used to quantitatively validate the potentiation effect of IL34 on BMP2 activation of SMAD1/5 phosphorylation. Co-additions of 25 or 50 ng/mL of IL34 increased significantly the phosphorylation of SMAD1/5 induced by the addition of BMP2 at 10 ng/mL. **p<0.01, ***p<0.001. (G) Western blot analysis of SMDA1/5 as described in B with a single concentration of IL34 (20 ng/mL) in combination with gradual quantities of IL34 (5, 10, 20, 40 and 80 ng/mL). (H) Western blot analysis of MCSFR phosphorylation expressed in HEK293 transfected cells in the presence or absence of BMP2 (10 ng/mL), IL34 (20 ng/mL) or in combination (BMP2+IL34, -) plus NOGGIN (NOG) (200 ng/mL). Quantifications of all the Western blots presented in this figure are shown in . All experiments have done at least three times independently.

Article Snippet: Antibodies directed against human Smad1 (D59D7), human Smad2 (D43B4), anti Phospho-Smad1/5 (ser463/465) (41D10), anti-phospho-Smad2 (Ser465/467) (138D), β-Actin (8H10D10) and HRP-conjugated secondary antibodies were purchased from Cell Signalling (Ozyme, Saint Quentin Yvelines, France).

Techniques: Western Blot, Blocking Assay, Control, Concentration Assay, Activation Assay, Transfection