Journal: Oncogene

Article Title: Smad6 is a protein kinase X phosphorylation substrate and is required for HL-60 cell differentiation.

doi: 10.1038/sj.onc.1209436

Figure Lengend Snippet: Figure 2 Phosphorylation of Smad6 by protein kinase X (PrKX). (a) Recombinant PrKX protein was purified from S. cerevisae and Smad6 from E. coli and reaction cocktails containing the proteins were assembled containing 0.5 mCi [g-32P]ATP in kinase buffer. Reactions proceeded for 30 min at 301C and were terminated by addition of 5 Laemlli buffer and boiling for 5 min followed by analysis by sodium dodecyl sulfate-polyacrylamide.gel electrophor- esis (SDS-PAGE) and autoradiography of reaction products. The arrows indicate the migration of PrKX and Smad6 proteins. (b) Similar reactions were performed with non-radiolabeled ATP and the reaction products were analysed by Western blotting. The blots were incubated with primary antibodies against Smad6 (goat polyclonal), or PrKX, phospho-serine, phopho-threonine and phospho-tyrosine (rabbit polyclonals). Immunoconjugates were visualized using either donkey-anti-goat or goat-anti-rabbit anti- bodies linked to horseradish peroxidase (HRP). (c) HL-60 cells stably transfected with a vector expressing His-tagged human Smad6 (HL60-pcSmad6-clone 10) were either untreated (0), or treated with PMA for 4 or 24 h. Cells were lysed and recombinant Smad6 protein purified using Ni-affinity chromatography. Bound proteins were eluted by imidazole competition, concentrated and electrophoresed using SDS-PAGE. The proteins were transferred by semi-dry blotting and probed using either an a-Smad6 antibody or phospho-serine antibody followed by chemiluminescent detec- tion of immunoconjugates.

Article Snippet: For immunoprecipitation, a goat polyclonal antibody directed against Smad6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was conjugated to agarose using the SeizeX kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.

Techniques: Phospho-proteomics, Recombinant, SDS Page, Autoradiography, Migration, Western Blot, Incubation, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Chromatography, Semi Dry Blot

Journal: Oncogene

Article Title: Smad6 is a protein kinase X phosphorylation substrate and is required for HL-60 cell differentiation.

doi: 10.1038/sj.onc.1209436

Figure Lengend Snippet: Figure 1 Co-immunoprecipitation of proteins from COS7B and HL-60 cells. (a) COS7B cells were transfected with vector pFLAG- CMV-2-PrKX by itself, or in combination with vector pcDNA3.1nV5/regulatory subunit I alpha of protein kinase A (PKAr1a), pcDNA3.1nV5/Smad6 or pcDNA3.1nV5/T54. Cells were recovered and immunoprecipitations performed using anti- Flag-M2 affinity resin. Recovered proteins were subjected to Western blotting analysis using either anti-V5 (upper panel) or anti-protein kinase X (PrKX) (lower panel) antibodies and horseradish peroxidase (HRP)-labeled secondary antibodies. (b) Native HL-60 cells were either untreated (c), or treated with 10 nM phorbol 12-myristate 13-acetate (PMA), 1 mM all-trans retinoic acid (ATRA) or 2.5 ng/ml transforming growth factor-b (TGF-b) for 24 h. Cells were recovered and cross-linked with 1% formaldehyde and then lysed using immunoprecipitation lysis buffer. For immunoprecipitation, a goat polyclonal antibody directed against Smad6 (Santa Cruz Biotechnology) or goat IgG (Sigma) was conjugated to agarose using the SeizeX kit (Pierce, Rockford, IL, USA) and the HL-60 lysates immunoprecipitated. Bound protein was analysed by Western blotting for the presence of PrKX using a polyclonal antibody directed against a synthetic PrKX peptide (upper panel), or for Smad6 using the goat anti-Smad6 antibody (lower panel).

Article Snippet: For immunoprecipitation, a goat polyclonal antibody directed against Smad6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was conjugated to agarose using the SeizeX kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Western Blot, Labeling, Lysis

Journal: Oncogene

Article Title: Smad6 is a protein kinase X phosphorylation substrate and is required for HL-60 cell differentiation.

doi: 10.1038/sj.onc.1209436

Figure Lengend Snippet: Figure 3 Definition of a protein kinase X (PrKX) phosphoryla- tion motif. (a) A putative peptide target for PrKX was synthesized with the amino-acid sequence PPVYSLQDFD (test); as a control, a peptide of similar composition and length, the fibronectin binding peptide RGDSPASSKP (cont.) was used. Phosphorylation reac- tions were assembled with 0.5 mCi [g-32P]ATP and 5–50 mg of each peptide were individually included in the reaction cocktail. Peptide reaction products were resolved using a Tris-tricine SDS gel with the separating gel being of 16.5% polyacrylamide (19:1 acrylami- de:bisacrylamide ratio) and 6 M urea. Separation was achieved by the application of 105 V for 16 h. Phosphorylated peptides were visualized by direct exposure of the wet gel to X-ray film. For the visualization of PrKX autophosphorylation, the same reactions were separated by sodium dodecyl sulfate-polyacrylamide.gel electrophoresis (SDS-PAGE) in 10% polyacrylamide gels (37.5:1 acrylamide:bisacrylamide ratio), the gels were dried and then exposed to X-ray film. (b) Serine435 of human Smad6 was converted to an alanine by site-directed mutagenesis, and the resultant protein purified from E. coli. Wild-type (wt) and mutant (mut) Smad6 were then assayed for phosphorylation by PrKX by incubation in kinase buffer containing radiolabeled ATP. Reaction products were separated by SDS-PAGE and visualized by autoradiography. In addition, Western blotting (lower panel) of resolved reaction products was performed using a polyclonal antibody directed against Smad6.

Article Snippet: For immunoprecipitation, a goat polyclonal antibody directed against Smad6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was conjugated to agarose using the SeizeX kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.

Techniques: Synthesized, Sequencing, Control, Binding Assay, Phospho-proteomics, SDS-Gel, Polyacrylamide Gel Electrophoresis, SDS Page, Mutagenesis, Incubation, Autoradiography, Western Blot

Journal: Oncogene

Article Title: Smad6 is a protein kinase X phosphorylation substrate and is required for HL-60 cell differentiation.

doi: 10.1038/sj.onc.1209436

Figure Lengend Snippet: Figure 4 Subcellular localization of protein kinase X (PrKX) and Smad6 during macrophage differentiation of HL-60 cells. HL-60 cells were either untreated or treated with phorbol 12-myristate 13- acetate (PMA) for 6 or 24 h. Total protein was collected by lysing pellets in 1 Laemmli buffer, cytoplasmic and nuclear compart- ments were separated by detergent disruption of the cell membrane and subsequent recovery of nuclear pellets. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide.gel electrophoresis (SDS-PAGE) and analysed by Western blotting using antibodies against PrKX, Smad6 and a loading standard (actin for the total and cytoplasmic fractions and histone for the nuclear fraction).

Article Snippet: For immunoprecipitation, a goat polyclonal antibody directed against Smad6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was conjugated to agarose using the SeizeX kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.

Techniques: Disruption, Membrane, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot

Journal: Oncogene

Article Title: Smad6 is a protein kinase X phosphorylation substrate and is required for HL-60 cell differentiation.

doi: 10.1038/sj.onc.1209436

Figure Lengend Snippet: Figure 5 Electrophoretic mobility shift assays (EMSAs). Double- stranded oligonucleotide probes corresponding to regions of the Hex (HexA), Id2 and osteopontin (OPN) promoters were labeled with 32P and used in EMSA reactions. Protein cocktails were assembled containing either in the presence or absence of phosphorylated Smad6 as indicated by the or þ above each lane. Promoter binding reactions were assembled in the presence or absence of nuclear extracts (NucEx) from HL-60 cells either untreated or treated with phorbol 12-myristate 13-acetate (PMA) for 24 h (C or P, respectively, as indicated above each lane) and the reactions incubated for 30 min at room temperature. The reaction products were then resolved by PAGE in non-denaturing gels followed by drying of the gel and autoradiography.

Article Snippet: For immunoprecipitation, a goat polyclonal antibody directed against Smad6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was conjugated to agarose using the SeizeX kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.

Techniques: Electrophoretic Mobility Shift Assay, Labeling, Binding Assay, Incubation, Autoradiography

Journal: Oncogene

Article Title: Smad6 is a protein kinase X phosphorylation substrate and is required for HL-60 cell differentiation.

doi: 10.1038/sj.onc.1209436

Figure Lengend Snippet: Figure 6 Chromatin immunoprecipitation (ChIP) and gene expression analysis in HL-60 cells. (a) HL-60 cells were untreated or treated with phorbol 12-myristate 13-acetate (PMA) for 6 or 24 h. Proteins were cross-linked with 1% paraformaldehyde and chromatin was collected and sheared by sonication. Immunopre- cipitates were collected with a goat polyclonal antibody to Smad6 followed by protein A–agarose binding. For the mock precipita- tion, equal amounts of HL-60 chromatin were combined and incubated in the absence of the primary Smad6 antibody; all other manipulations were identical to those performed on the test samples. Input chromatin (‘input’ lane) was obtained as the DNA- containing supernatant of the ‘mock’ immunoprecipitation reac- tion. DNA was recovered from the precipitates and analysed by PCR using primers encompassing the Smad/transforming growth factor-b (TGF-b)/bone morphogenetic protein (BMP)-responsive elements of the human osteopontin Id2, and Hex promoters. Chromatin immunoprecipitation (ChIP) assays were repeated twice with similar results obtained each time; the results of one such repetition are shown here. (b) Total RNA was collected from HL- 60 cells either untreated or exposed to 10 nM PMA for 6 or 24 h. cDNA was prepared by oligo-dT-primed first-strand synthesis followed by PCR using primers specific for OPN, Id2, Hex, PrKX, cd11b or glyceraldehyde 3-phosphate dehydrogenase (gapdh) mRNAs.

Article Snippet: For immunoprecipitation, a goat polyclonal antibody directed against Smad6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was conjugated to agarose using the SeizeX kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.

Techniques: Chromatin Immunoprecipitation, Gene Expression, Sonication, Binding Assay, Incubation, Immunoprecipitation

Journal: Oncogene

Article Title: Smad6 is a protein kinase X phosphorylation substrate and is required for HL-60 cell differentiation.

doi: 10.1038/sj.onc.1209436

Figure Lengend Snippet: Figure 7 Inhibition of phorbol 12-myristate 13-acetate (PMA)- induced cell attachment and spreading by short interfering RNA (siRNA) mediated inhibition of Smad6 and PrKX. Double- stranded DNA oligonucleotides targeting Smad6 (Smad6-A, Smad6-B) and PrKX (PrKX-A) were cloned into vector pSilencer 2.0 vectors and the vectors were further modified to contain an enhanced green fluorescent protein (EGFP) expression cassette. Vectors were electroporated into HL-60 cells, the cells were allowed to recover for 6 h and then were induced with 10 nM PMA for 16 h. Unattached and attached and spread cell fractions were collected and analysed for GFP expression, a marker for successful transfection of an individual cell, using fluorescence microscopy. EGFP expression was determined for 800 cells per data point.

Article Snippet: For immunoprecipitation, a goat polyclonal antibody directed against Smad6 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was conjugated to agarose using the SeizeX kit (Pierce, Rockford, IL, USA) according to the manufacturer’s instructions.

Techniques: Inhibition, Cell Attachment Assay, Small Interfering RNA, Clone Assay, Plasmid Preparation, Expressing, Marker, Transfection, Microscopy

Journal: The Febs Journal

Article Title: The m6A demethylase FTO promotes C/ EBPβ ‐ LIP translation to perform oncogenic functions in breast cancer cells

doi: 10.1111/febs.70033

Figure Lengend Snippet: FTO knockdown has a limited effect on mRNA translation. (A) Fold change in translation efficiency upon FTO knockdown in MDA‐MB‐231 cells is displayed, color coded by FDR. Significantly regulated genes above threshold are indicated by gene symbol. (B) RT‐qPCR analysis of DICER mRNA expression in MDA‐MB‐231 cells with FTO knockdown (shFTO1 or shFTO2) versus shRNA‐scrambled (scr) control. Significance was determined by one‐way ANOVA with Dunnett's post hoc test (mean ± SEM, n = 3 biological replicates). (C) Immunoblots showing no change in DICER protein expression upon knockdown of FTO (shFTO1 or shFTO2) in MDA‐MB‐231 versus scrambled control (SCR). β‐Actin was used as loading control. Quantification is shown on the right. Significance was determined by one‐way ANOVA with Dunnett's post hoc test (mean ± SEM, n = 3 biological replicates). (D) RT‐qPCR analysis of COL1A1 and SMAD6 mRNA expression in MDA‐MB‐231 upon FTO knockdown (shFTO1 or shFTO2) versus shRNA‐scrambled (scr) control. Significance was determined by one‐way ANOVA with Dunnett's post hoc test (mean ± SD, n = 3 biological replicates). (E) Immunoblot analysis of COL1A1, SMAD6, and DICER protein expression in MDA‐MB‐231 upon FTO knockdown (shFTO1 or shFTO2) versus shRNA‐scrambled (scr) control, and β‐Actin as loading control. 5 days and 20 days refer to days in cell culture after transduction with shRNA retroviral vector. (F) Subsequence abundance analysis of codons at the +15 position relative to the ribosome protected fragment (RPF) 5′‐end. All transcripts in the coding frame are considered for this analysis. No significant enrichment or depletion of codons can be seen, indicating no obvious shortages of amino acids causing ribosome stalls/halts.

Article Snippet: The following primary antibodies were used for detection: FTO: NB110‐60935, Novus Biologicals, Abingdon, UK (1:1666); β‐Actin: 691001, MP Bio, Heerlen, The Netherlands (1:5000); C/EBPβ: ab32358, Abcam, Cambridge, UK (1:1000); WTAP: #56501 (1:1000), Cell Signaling Technology, Leiden, The Netherlands; COL1A1: #72026 (1:1000), Cell Signaling Technology, Leiden, The Netherlands; SMAD6: NB100‐56440, Novus Biologicals, Abingdon, UK (1:1000), FLAG‐M2: F‐3165, Sigma Aldrich, Merck, Dordrecht, The Netherlands (1:1000).

Techniques: Knockdown, Quantitative RT-PCR, Expressing, shRNA, Control, Western Blot, Cell Culture, Transduction, Retroviral, Plasmid Preparation

Journal: Oncoscience

Article Title: Bone morphogenetic protein 4 (BMP-4) and epidermal growth factor (EGF) inhibit metalloproteinase-9 (MMP-9) expression in cancer cells

doi:

Figure Lengend Snippet: (A) MMP-9 mRNA expression in mock-transfected HT1080 cells or HT1080 cells transfected with an expression vector encoding a flagged Smad6 (pCS2-Smad6-Flag). MMP-9 expression was measured 16h after transfection. The lower panel represents the control Western blot gels showing de novo expression of Smad6 in transfected cells. GAPDH and actin were used as loading and specificity controls for RT-PCR and Western blot analyses respectively. In (B), a zymogram showing MMP-9 in the supernatant of HT1080 transfected cells. Data are representative of at least three independent experiments.

Article Snippet: The vector encoding Smad6 (pCS2-Flag-Smad6) was obtained from Addgene (Cambridge, MA).

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Life Science Alliance

Article Title: Tissue-specific I-Smad mechanisms revealed by structure–function analysis in Drosophila

doi: 10.26508/lsa.202503445

Figure Lengend Snippet: (A, B) AlphaFold3 structural predictions of MH1 domains. The β-hairpin region is shown in yellow. Remaining MH1 is shown in blue. (A) Smad3 MH1 domain showing DNA-binding region and predicted interaction. (B) Dad, Smad6, and Smad7 MH1 domains. (C) Representative wings from females expressing indicated construct in wing primordium. The scale bar represents 1 mm. (D, E) Transgenic control ( TwitGFP , OK371-Gal4/+ ) compared with OK371-Gal4 –driven overexpression of UAS-Dad , UAS-Smad6 , UAS-Smad7L , and UAS-Smad7S . (D) Motor neurons in ventral nerve cord triple-labeled for Elav (blue), TwitGFP (green), and pMad (red). The scale bar represents 20 microns. (E) Quantification of pMad and TwitGFP intensity normalized to the genetic background control. Bars represent the mean ± SEM. Each dot represents mean data from motor neurons of one CNS. Statistical comparisons were performed using Dunn’s test with the Bonferroni correction. **** P < 0.0001, ** P < 0.01. Number of brains analyzed for pMad in the order shown in the graph: 52, 24, 25, 20, and 28. Number of brains analyzed for TwitGFP in the order shown in the graph: 53, 27, 22, 19, and 26.

Article Snippet: CS2 Smad6 , N/A , CS2 Smad6 was a gift from Joan Massague (plasmid #14960; https://n2t.net/addgene:14960 ; RRID:Addgene_14960; Addgene) ( ) , Source of Smad6 CDS.

Techniques: Binding Assay, Expressing, Construct, Transgenic Assay, Control, Over Expression, Labeling

Journal: Endocrinology

Article Title: Inhibitor of Differentiation (Id) genes are expressed in the steroidogenic cells of the ovine ovary and are differentially regulated by members of the Transforming Growth Factor (TGF)-? family

doi: 10.1210/en.2009-0914

Figure Lengend Snippet: List of primary, secondary and tertiary antibodies used for immunohistochemistry (IH) and immunofluorescence (IF). Secondary antibody; 1) Biotinylated Goat anti-Rabbit IgG (Dako, Glostrup, Denmark) 1:500, 2) Peroxidase Goat anti-Rabbit (Dako) 1:200. Tertiary antibody; A) Streptavidin Alexafluor 546 (s-11225 Invitrogen, Paisley, UK) 1:200, B) Tyramide Fluorescein (Perkin Elmer LAS, Inc. MA, USA) 1:50.

Article Snippet: Smad 6 , Polyclonal Rabbit (IMG-555), Imgenex Corp, Cambridge BioScience, Cambs, UK , IH 1:100 IF 1:600 , 2 , B.

Techniques: Immunohistochemistry, Immunofluorescence