Journal: PLoS ONE

Article Title: Galnt1 Is Required for Normal Heart Valve Development and Cardiac Function

doi: 10.1371/journal.pone.0115861

Figure Lengend Snippet: (A–D) Confocal images and quantification of phospho-histone H3 (pH3) and Ki-67 proliferation markers in developing valves at E11.5 and E12.5. (A) E11.5 OFT cushion tissue (pH3, green; myocardial marker MF20, red; n = 4). (B) E11.5 OFT cushion tissue (Ki-67, green; cell surface marker lectin WGA, red; n = 3). (C) E12.5 OFT cushion tissue (pH3, green; myocardial marker MF20, red; n = 3). (D) E12.5 OFT cushion tissue (Ki-67, green; cell surface marker lectin WGA, red; n = 3). White dash lines in lower magnification in A–D highlight developing cushion area. Higher magnification views are shown to the right of each image. Percentage of nuclei (Nu; blue) positive for pH3 or Ki-67 in wild type samples was set to 1 and relative differences in Galnt1 nulls is graphed to the right of each image. Western blotting reveals an increase in phosphorylation of (E) Smad1/5 (BMP) and (F) MAPK (ERK1/2), (G) decreased EGFR in Galnt1 null OFT samples relative to wild type at E11.5 (n = 4), and E12.5 (n = 6) while remaining unchanged in E10.5 OFT (n = 3) and in E14.5 AoV (n = 3). Samples were normalized to total Smad1, total MAPK and α-Tubulin (α-Tub). (H) Expression of Bmp/Smad genes was examined by qPCR in E12.5 OFT cushion (OFTC) tissues (triplicates, n = 5) isolated by laser-capture microdissection (LCM). While expression of Bmp2 and Bmpr2 remains unchanged, gene expression of Msx1 (a Bmp2 downstream target gene) is significant increased, and Smad6 expression (an inhibitor of BMP signaling) is greatly reduced. Student’s t-test was used to calculate P-valves. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: A, B, C, D = 100 μm for low magnification and 10 μm for higher magnification view.

Article Snippet: For Smad, MAPK and EGFR western blot analyses, membranes were blocked with 5% BSA-TBST and probed with phospho-Smad1/5 and phospho-MAPK (Erk1/2), and total-Smad1 and MAPK antibodies (Cell Signaling #9516P, #4376, #6944, #9102, respectively; rabbit monoclonal for first 3 antibodies and rabbit polyclonal for the fourth antibody; 1:1000), or EGFR antibody (Cell Signaling #4267; rabbit polyclonal antibody; 1:1000) and α-Tubulin antibody (Cell Signaling #2125; rabbit monoclonal antibody; 1:1000).

Techniques: Marker, Western Blot, Phospho-proteomics, Expressing, Isolation, Laser Capture Microdissection, Gene Expression

Journal: Oncotarget

Article Title: BMPR2 promotes invasion and metastasis via the RhoA-ROCK-LIMK2 pathway in human osteosarcoma cells

doi: 10.18632/oncotarget.17382

Figure Lengend Snippet: (A) Cytoskeletal assay of 143B and U2OS cells was visualized by confocal microscopy. Representative images were shown. Cell nuclei were stained with Hoechst33342. Scale bar represents 25 μm or 10 μm. (B) BMPR2-silencing attenuates p-LIMK expression through the Smad-independent pathway. (C) Smad1 and Smad5 are not required for BMPR2-depleted down-regulation of p-LIMK2. (D) GTPase activation assay validated the changes of RhoA in U2OS cells. (E) Interaction of LIMK2 with BMPR2 in U2OS cells. U2OS cells were transfected with Flag-BMPR2 and Flag-control. Cell lysates were immunoprecipitated with Flag-beads, and immunoblotted with anti-BMPR2 (top panel) or anti-LIMK2 antibodies (bottom panel).

Article Snippet: Antibodies against E-cadherin (no. 3195), N-cadherin (no.13116), Vimentin (no. 5741), ZEB1 (no. 3396), p-cofilin (no.3313), cofilin (no.5175), p-GSK3β (no.9322), GSK3β (no.12456), p-Smad1/5 (no. 9516), smad1 (no.6944) and smad5 (no.12534) were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Confocal Microscopy, Staining, Expressing, Activation Assay, Transfection, Control, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Arginine methylation of R81 in Smad6 confines BMP-induced Smad1 signaling

doi: 10.1016/j.jbc.2021.100496

Figure Lengend Snippet: R81-methylated Smad6 binds to Smad1. A , Smad6 R81 methylation was required for binding Smad1 in vitro . GST-tagged Smad6 WT or R81A mutant was methylated by recombinant PRMT1 in vitro in the presence of SAM and then incubated with FLAG–Smad1–conjugated Sepharose beads. The bound and the flow-through fractions were assessed by immunoblotting (IB). B , R81 methylation was required for Smad6 binding to Smad1 in vivo . HaCaT cells were transfected with FLAG-tagged Smad6 WT or R81A mutant, treated with BMP4 and subjected to immunoprecipitation (IP) with either anti-FLAG antibody or anti-GFP antibody as control. The presence of Smad1 and pSmad1 in the immunocomplexes was detected by immunoblotting. C , endogenous Smad1 interacted with endogenous R81-methylated Smad6. HaCaT cells were treated with BMP4, and endogenous Smad1 was immunoprecipitated with anti-Smad1 antibodies. The coprecipitated Smad6 and Smad1 and the R81 methylation of Smad6 were assessed by immunoblotting. D , R81-methylated Smad6 preferred the active phosphorylated Smad1 for binding. GST–Smad6 was methylated by PRMT1 in the presence of SAM and incubated with FLAG-tagged pSmad1-conjugated beads that had been treated with CIP as indicated. The beads were washed and subjected to Western blot (WB) analysis. E , BMP4 induced R81 methylation of Smad6 in HaCaT cells. HaCaT cells were subjected to BMP4 treatment followed by WB analysis of R81-methylated Smad6 and Smad1. F , the R81A mutant enhanced interaction between Smad6 N-terminal and C-terminal domains. Hemagglutinin (HA)-tagged Smad6 1–330 , either WT or R81A mutant, was coexpressed with FLAG-tagged Smad6 331–495 . The interaction between Smad6 1–330 and Smad6 331–495 was assessed by co-IP. G and H , PRMT1 silencing enhanced the interaction between Smad6 N-terminal and C-terminal domains without further enhancement because of the R81A mutation. Panel G shows the efficient silencing of PRMT1 expression. The 293T cells stably expressing control shRNA or shRNA targeting PRMT1 were assessed by immunoblotting. In panel H , HA-tagged Smad6 1–330 , either WT or the R81A mutant, was coexpressed with FLAG-tagged Smad6 331–495 in 293T cells stably expressing control shRNA or shRNA targeting PRMT1. The interaction between Smad6 1–330 and Smad6 331–495 was assessed by co-IP. All experiments were independently repeated at least three times. AP, affinity purification; CIP, calf intestinal alkaline phosphatase; PRMT1, protein arginine methyltransferase 1; pSmad1, phosphorylated Smad1; R81, arginine 81; SAM, S-adenosyl methionine.

Article Snippet: Rabbit polyclonal antibodies against asymmetric di-methyl R81-Smad6 were generated by New England Peptide LLC as described previously ( ); anti-Smad6 (cat# 9519), anti-Smad1/5 (cat# 9516), anti-Smad1 (cat# 6944), and anti-PRMT1 antibodies (cat# 2449) were purchased from Cell Signaling Technology; anti-Smad7 (cat# sc-11392) anti-Smad4 (cat# sc-7966), anti-GFP (cat# sc8334), anti-HDAC1 (cat# sc7872), anti-GST (cat# sc-138) and anti-α-tubulin antibodies (cat# sc-5286) were purchased from Santa Cruz Biotechnology; anti-FLAG antibodies (cat# F3165) and anti-FLAG-M2 affinity agarose (cat# A2220) were purchased from Sigma-Aldrich; Anti-Myc antibodies (cat# 05-724) were purchased from Millipore; and anti-HA (cat# 90515 BioLegend) antibodies (cat# 901515) were from BioLegend; anti-GAPDH (cat# GTX627408) and Anti-BMPRIAca antibodies (cat# GTX113140) were from GeneTex.

Techniques: Methylation, Binding Assay, In Vitro, Mutagenesis, Recombinant, Incubation, Western Blot, In Vivo, Transfection, Immunoprecipitation, Co-Immunoprecipitation Assay, Expressing, Stable Transfection, shRNA, Affinity Purification

Journal: The Journal of Biological Chemistry

Article Title: Arginine methylation of R81 in Smad6 confines BMP-induced Smad1 signaling

doi: 10.1016/j.jbc.2021.100496

Figure Lengend Snippet: R81 methylation enables Smad6 to disrupt Smad1–Smad4 complex formation. A , the 293T cells were transfected with FLAG-tagged Smad6 WT, the R81A mutant or the R81K mutant, MYC-tagged Smad4, FLAG-tagged Smad1, and HA-tagged constitutively active BMPRIA (BMPRIAca). The interaction between Smad1 and Smad4 was assessed by co-IP with anti-MYC antibody (Smad4) followed by the Western blot analysis of Smad1 and pSmad1. B , HaCaT cells were transfected with increasing amounts of FLAG-tagged Smad6 WT or the R81A mutant and treated with BMP4. The interaction between Smad1 and Smad4 was assessed by co-IP with anti-Smad4 antibody followed by Western Blot. Anti-GFP antibody serves as control antibody for IP. All experiments were independently repeated at least three times. co-IP, coimmunoprecipitation; IP, immunoprecipitation; pSmad1, phosphorylated Smad1; R81, arginine 81.

Article Snippet: Rabbit polyclonal antibodies against asymmetric di-methyl R81-Smad6 were generated by New England Peptide LLC as described previously ( ); anti-Smad6 (cat# 9519), anti-Smad1/5 (cat# 9516), anti-Smad1 (cat# 6944), and anti-PRMT1 antibodies (cat# 2449) were purchased from Cell Signaling Technology; anti-Smad7 (cat# sc-11392) anti-Smad4 (cat# sc-7966), anti-GFP (cat# sc8334), anti-HDAC1 (cat# sc7872), anti-GST (cat# sc-138) and anti-α-tubulin antibodies (cat# sc-5286) were purchased from Santa Cruz Biotechnology; anti-FLAG antibodies (cat# F3165) and anti-FLAG-M2 affinity agarose (cat# A2220) were purchased from Sigma-Aldrich; Anti-Myc antibodies (cat# 05-724) were purchased from Millipore; and anti-HA (cat# 90515 BioLegend) antibodies (cat# 901515) were from BioLegend; anti-GAPDH (cat# GTX627408) and Anti-BMPRIAca antibodies (cat# GTX113140) were from GeneTex.

Techniques: Methylation, Transfection, Mutagenesis, Co-Immunoprecipitation Assay, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Arginine methylation of R81 in Smad6 confines BMP-induced Smad1 signaling

doi: 10.1016/j.jbc.2021.100496

Figure Lengend Snippet: Smad6 R81 methylation is required for the inhibition of BMP4-induced Smad1 nuclear translocation. A , HaCaT cells were transiently transfected with FLAG-tagged Smad6 WT or the R81A mutant or the R81K mutant, treated with BMP4 or vehicle for 1 h and then subjected to immunostaining with anti-Smad1 primary antibodies and Alexa fluor 594–conjugated secondary antibody. Nuclei were imaged based on DAPI staining. The arrows indicate transfected cells. Note the weak nuclear Smad1 staining in BMP4-treated cells expressing WT Smad6 ( white arrows ) compared with nontreated cells or cells expressing mutant Smad6 ( yellow arrows ). See Fig. S1 A for larger fields of view. B , HaCaT cells stably expressing Smad6 WT, the R81A mutant, or GFP as control were treated with BMP4 or vehicle for 2 h and subjected to immunostaining with anti-Smad1 antibody. Representative images are shown in Fig. S1 B . The ratio of nuclear/cytosolic Smad1 was quantified in 100 to 200 cells per group using ImageJ (mean ± SEM). ∗ p < 0.05, ∗∗ p < 0.01 versus vehicle. C – F , HaCaT cells stably expressing Smad6 WT, Smad6 R81A, or GFP as control were treated with BMP4 for the indicated periods of time and subjected to cellular fractionation. The cytosolic and nuclear fractions were analyzed by immunoblotting using the indicated antibodies, where HDAC1 and GAPDH serve as loading controls. Immunoblot density was quantified for those in panels E and F using ImageJ. G and H , Smad6 WT, but not the R81 mutant, abrogates BMP-responsive gene expression. HaCaT cells stably expressing Smad6 WT, Smad6 R81A, or GFP as control were treated with BMP4 or vehicle for 2 h, and expression of ID1 ( G ) and ID3 ( H ) was measured by RT-qPCR (mean ± SEM). ∗ p < 0.05, ∗∗ p < 0.01 versus vehicle. All experiments were independently repeated at least three times. I , working model: PRMT1 methylates Smad6 at R81, which facilitates Smad6/pSmad1 interaction that competes with Smad1–Smad4 complex formation to inhibit Smad1-mediated BMP signaling. BMP, bone morphogenetic protein; PRMT1, protein arginine methyltransferase 1; R81, arginine 81.

Article Snippet: Rabbit polyclonal antibodies against asymmetric di-methyl R81-Smad6 were generated by New England Peptide LLC as described previously ( ); anti-Smad6 (cat# 9519), anti-Smad1/5 (cat# 9516), anti-Smad1 (cat# 6944), and anti-PRMT1 antibodies (cat# 2449) were purchased from Cell Signaling Technology; anti-Smad7 (cat# sc-11392) anti-Smad4 (cat# sc-7966), anti-GFP (cat# sc8334), anti-HDAC1 (cat# sc7872), anti-GST (cat# sc-138) and anti-α-tubulin antibodies (cat# sc-5286) were purchased from Santa Cruz Biotechnology; anti-FLAG antibodies (cat# F3165) and anti-FLAG-M2 affinity agarose (cat# A2220) were purchased from Sigma-Aldrich; Anti-Myc antibodies (cat# 05-724) were purchased from Millipore; and anti-HA (cat# 90515 BioLegend) antibodies (cat# 901515) were from BioLegend; anti-GAPDH (cat# GTX627408) and Anti-BMPRIAca antibodies (cat# GTX113140) were from GeneTex.

Techniques: Methylation, Inhibition, Translocation Assay, Transfection, Mutagenesis, Immunostaining, Staining, Expressing, Stable Transfection, Cell Fractionation, Western Blot, Quantitative RT-PCR