Journal: PLoS ONE

Article Title: Protein Kinase D1 Maintains the Epithelial Phenotype by Inducing a DNA-Bound, Inactive SNAI1 Transcriptional Repressor Complex

doi: 10.1371/journal.pone.0030459

Figure Lengend Snippet: A: The amino-acids surrounding serine 11 in SNAI1 form a PKD consensus motif as it was described for S82 of Hsp27 and S978 of SSH1L. B: PKD phosphorylates SNAI1 at S11 in an in vitro assay. Bacterially-expressed and purified GST (negative control), GST-SNAI1 or GST-SNAI1.S11A were incubated in a kinase reaction with purified active PKD1. Substrate phosphorylation was detected using the pMOTIF antibody, which recognizes the phosphorylated PKD motif in PKD substrates or with the novel pS11-SNAI1 antibody specifically generated for this site. Control blots were performed for protein input (anti-PKD1, anti-GST). C, D: HeLa cells were transfected with combinations of vector control, active PKD1 (PKD1.CA) and SNAI1 or SNAI1.S11A mutant as indicated. PKD-mediated phosphorylation of SNAI1 was detected using the pMOTIF (C) or the pS11-SNAI1 (D) antibodies. E, F: HeLa cells were transfected with combinations of vector control, active RhoA (RhoA.CA) and PKD1 or PKD1.KD mutant (E) or control shRNA and shRNA specific for PKD1/2 (F) as indicated and FLAG-tagged SNAI1. PKD-mediated phosphorylation of SNAI1 was detected using the pS11-SNAI1 antibody. Samples were also control-stained for SNAI1 and PKD1 expression using anti-FLAG or anti-PKD1 antibodies, respectively. Anti-GST control staining for RhoA.CA and GST control are depicted in . G: NMuMG cells were treated with TGFβ1 (10 ng/ml) for 48 hours. Total cell lysates were analyzed for phosphorylation of endogenous SNAI1 at S11 (anti-pS11-SNAI1) or PKD1 activity (anti-pS738/742-PKD) or total PKD1 expression (anti-PKD1) as indicated. H: NMuMG cells were treated with CID755673 (25 µM, 4 hr) or left untreated as indicated. Total cell lysates were analyzed for phosphorylation of endogenous SNAI1 at S11 (anti-pS11-SNAI1) or SNAI1 expression (anti-SNAI1).

Article Snippet: A rabbit polyclonal antibody specific for human SNAI1 phosphorylated at S11 (anti-pS11-SNAI1 antibody) was raised by 21 Century Biochemicals (Marlboro, MA) using Ac-FLVRKP[pS]DPNRKPC-amide and Ac-CFLVRKP[pS]DPNRKPN-amide peptides as antigens.

Techniques: In Vitro, Purification, Negative Control, Incubation, Phospho-proteomics, Generated, Control, Transfection, Plasmid Preparation, Mutagenesis, shRNA, Staining, Expressing, Activity Assay

Journal: PLoS ONE

Article Title: Protein Kinase D1 Maintains the Epithelial Phenotype by Inducing a DNA-Bound, Inactive SNAI1 Transcriptional Repressor Complex

doi: 10.1371/journal.pone.0030459

Figure Lengend Snippet: A: Immunofluorescence staining of NMuMG cells for endogenous PKD1 (anti-PKD1). The bar represents 10 µm. B: Immunofluorescence staining of NMuMG cells for S11-phosphorylated SNAI1 (anti-pS11-SNAI1) in absence or presence of competing phospho-S11-peptide and nuclei (DAPI). The bar represents 10 µm. C: HeLa cells were transfected as indicated and nuclear extracts were prepared and analyzed by Western blot for SNAI1 (anti-FLAG), pS11-SNAI1 (anti-pS11-SNAI1) and nucleolin (anti-nucleolin, loading control). D: NMuMG cells were transfected with GFP control, GFP-SNAI1, GFP-SNAI1.S11A or GFP-SNAI1.S11E mutants. Localization of GFP or GFP-tagged proteins was determined using immunofluorescence analysis (bar is 10 µm). E: NMuMG cells were transfected with FLAG-tagged wildtype SNAI1 or SNAI1.S11A mutant and GFP-tagged, active PKD1 (PKD1.CA) as indicated and localization of SNAI1 was determined by indirect immunofluorescence staining (anti-FLAG as primary antibody). The bar represents 10 µm.

Article Snippet: A rabbit polyclonal antibody specific for human SNAI1 phosphorylated at S11 (anti-pS11-SNAI1 antibody) was raised by 21 Century Biochemicals (Marlboro, MA) using Ac-FLVRKP[pS]DPNRKPC-amide and Ac-CFLVRKP[pS]DPNRKPN-amide peptides as antigens.

Techniques: Immunofluorescence, Staining, Transfection, Western Blot, Control, Mutagenesis

Journal: PLoS ONE

Article Title: Protein Kinase D1 Maintains the Epithelial Phenotype by Inducing a DNA-Bound, Inactive SNAI1 Transcriptional Repressor Complex

doi: 10.1371/journal.pone.0030459

Figure Lengend Snippet: A: Hek293T cells were transfected with vector control, SNAI1 or active PKD1 (PKD1.CA) and SNAI1 as indicated. SNAI1/DNA complexes were immunoprecipitated (anti-FLAG) after crosslinking and precipitates were analyzed by PCR for the SNAI1-bound E-cadherin promoter. B: NMuMG cells were transfected with vector control, SNAI1, SNAI1.S11A or SNAI1.S11E mutants. SNAI1/DNA complexes were immunoprecipitated (anti-FLAG) after crosslinking and precipitates were analyzed by PCR for the SNAI1-bound E-cadherin promoter. C: NMuMG cells were treated with CID755673 (25 µM, 1 hr) or left untreated. Phospho-S11-SNAI1/DNA complexes were immunoprecipitated (anti-pS11-SNAI1) after crosslinking and precipitates were analyzed by PCR for the pS11-SNAI1-bound E-cadherin promoter. In experiments depicted in A–C , a PCR for the E-cadherin promoter using the input DNA as well as a ChIP using IgG instead of the anti-FLAG antibody served as controls. D: Hek293T cells were transfected with vector control, SNAI1 or SNAI1.S11A mutant, active PKD1 (PKD1.CA) or both and E-cadherin promoter luciferase reporter and renilla reporter plasmids. Induced luciferase activity was measured. Error bars shown represent standard deviations. P values were acquired with the t test, using GraphPad software. Asterisks indicate statistical significance.

Article Snippet: A rabbit polyclonal antibody specific for human SNAI1 phosphorylated at S11 (anti-pS11-SNAI1 antibody) was raised by 21 Century Biochemicals (Marlboro, MA) using Ac-FLVRKP[pS]DPNRKPC-amide and Ac-CFLVRKP[pS]DPNRKPN-amide peptides as antigens.

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation, Mutagenesis, Luciferase, Activity Assay, Software

Journal: PLoS ONE

Article Title: Protein Kinase D1 Maintains the Epithelial Phenotype by Inducing a DNA-Bound, Inactive SNAI1 Transcriptional Repressor Complex

doi: 10.1371/journal.pone.0030459

Figure Lengend Snippet: Tissue microarrays (TMAs) including 10 normal breast tissue samples, 40 invasive ductal carcinoma of the breast and 10 metastatic invasive ductal carcinoma samples from lymph nodes were H&E stained or analyzed for the expression of active PKD (anti-pY95-PKD), S11-phosphorylated SNAI1 (anti-pS11-SNAI1) and total SNAI1 (anti-SNAI1). Representative pictures of normal ( A–D ) and 3 tumor tissues ( E–P ) are depicted. Numbers indicate the position of the tissue on the TMA. The asterisk (sample #10) indicates tumor tissue form a region adjacent to the normal tissue (same patient). Inserts show enhanced area.

Article Snippet: A rabbit polyclonal antibody specific for human SNAI1 phosphorylated at S11 (anti-pS11-SNAI1 antibody) was raised by 21 Century Biochemicals (Marlboro, MA) using Ac-FLVRKP[pS]DPNRKPC-amide and Ac-CFLVRKP[pS]DPNRKPN-amide peptides as antigens.

Techniques: Staining, Expressing