Journal: Biochimica et biophysica acta

Article Title: Symplekin, a polyadenylation factor, prevents MOZ and MLL activity on HOXA9 in hematopoietic cells.

doi: 10.1016/j.bbamcr.2013.08.013

Figure Lengend Snippet: Fig. 1. Symplekin is associated with MOZ and MLL. (A) Symplekin interacts with MOZ in KG1 cells. IP was performed with anti-MOZ, then immunoprobed with anti-MOZ or anti-Sympk. (B) Symplekin interacts with MLL. IP was performed with anti-MLL, then immunoprobed with anti-MLL or anti-Sympk. (C) Symplekin interacts with MOZ in HEK293T cells. IP was performed with anti-MOZ, then immunoprobed with anti-MOZ or anti-Sympk. (D) Flag-Symplekin interacts with c-Myc-MOZ. Flag-Sympk or c-Myc-MOZ vectors were transfected into HEK293T cells. IP were performed with anti-Flag or anti-c-Myc, then immunoprobed as indicated. (E, F, G) Symplekin interacts directly with MOZ but not with MLL. As indicated, in vitro pulldown assays were performed with Flag-Sympk, Flag-Nter-Sympk, c-Myc-MOZ (E,F) or MLL (G), biotinylated (biot) or not (unbiot), produced by in vitro translation in reticu- locytes lysates. Pulldown of biotinylated proteins was performed with neutravidin-coated agarose beads and protein interactions were revealed by SDS-PAGE and immunoblotted with anti-Flag, Streptavidin, anti-c-Myc.

Article Snippet: MOZ (a mouse monoclonal antibody directed against residues 856–870 of MOZ (IGBMC, Illkirch, France)), Symplekin (Becton–Dickinson), MLL-C (Upstate Biotechnology), c-Myc (9E10, Santa Cruz Biotechnology), Acetyl Lysine (Cell Signaling Technology) specific antibodies associated with protein G agarose beads, were used for immunoprecipitating proteins with gentle shaking at 4 °C overnight.

Techniques: Transfection, In Vitro, Produced, SDS Page

Journal: Biochimica et biophysica acta

Article Title: Symplekin, a polyadenylation factor, prevents MOZ and MLL activity on HOXA9 in hematopoietic cells.

doi: 10.1016/j.bbamcr.2013.08.013

Figure Lengend Snippet: Fig. 2. (A) MOZ interacts with CPSF100 in KG1 cells. IP was performed with anti-MOZ, then immunoprobed with anti-MOZ or anti-CPSF100. (B) Symplekin is acetylated. A first IP was performed with anti-Sympk, eluated, then a second IP was performed with anti-acetylated lysine (anti-acK) and immunoprobed with anti-Sympk.

Article Snippet: MOZ (a mouse monoclonal antibody directed against residues 856–870 of MOZ (IGBMC, Illkirch, France)), Symplekin (Becton–Dickinson), MLL-C (Upstate Biotechnology), c-Myc (9E10, Santa Cruz Biotechnology), Acetyl Lysine (Cell Signaling Technology) specific antibodies associated with protein G agarose beads, were used for immunoprecipitating proteins with gentle shaking at 4 °C overnight.

Techniques:

Journal: Biochimica et biophysica acta

Article Title: Symplekin, a polyadenylation factor, prevents MOZ and MLL activity on HOXA9 in hematopoietic cells.

doi: 10.1016/j.bbamcr.2013.08.013

Figure Lengend Snippet: Fig. 3. Symplekin co-localizes with MOZ. (A) MOZ was stained with anti-MOZ and Symplekin with anti-Sympk in KG1 cells. Nuclei were counterstained with DAPI. The merge is the overlay of both images (scale bar: 9 µm). (B) MOZ was stained with anti-MOZ and Symplekin with anti-Sympk in HEK293T cells. Nuclei were counterstained with DAPI. The merge is the overlay of both images (scale bar: 9 µm). (C) Flag-Sympk or c-Myc-MOZ vectors were transfected into HEK293T cells. c-Myc-MOZ was stained with anti-c-Myc and Flag-Symplekin with anti-Flag. Nuclei were counterstained with DAPI. The merge is the overlay of both images (scale bar: 6 µm).

Article Snippet: MOZ (a mouse monoclonal antibody directed against residues 856–870 of MOZ (IGBMC, Illkirch, France)), Symplekin (Becton–Dickinson), MLL-C (Upstate Biotechnology), c-Myc (9E10, Santa Cruz Biotechnology), Acetyl Lysine (Cell Signaling Technology) specific antibodies associated with protein G agarose beads, were used for immunoprecipitating proteins with gentle shaking at 4 °C overnight.

Techniques: Staining, Transfection

Journal: Biochimica et biophysica acta

Article Title: Symplekin, a polyadenylation factor, prevents MOZ and MLL activity on HOXA9 in hematopoietic cells.

doi: 10.1016/j.bbamcr.2013.08.013

Figure Lengend Snippet: Fig. 5. Symplekin affects HOXA9 protein level in KG1 cells. (A) Symplekin impacts HOXA9 expression. Cells were transfected with siCtrl, siMOZ or siSympk. HOXA9 expression was measured 24 h after transfection by RQ-PCR (bar graphs: error bars represent standard deviation) (n = 5). (B) Symplekin affects HOXA9 protein level. Cells were transfected with siCtrl, siMOZ or siSympk. HOXA9 expression was measured 24 h after transfection by immunoblotting performed with anti-Sympk, anti-MOZ, anti-HOXA9 or anti-HSC70. One independent experiment out of three is shown (left panel). Quantitation of HOXA9 and Symplekin proteins from three independent experiments are also shown (bar graphs: error bars represent standard deviation) (right panel).

Article Snippet: MOZ (a mouse monoclonal antibody directed against residues 856–870 of MOZ (IGBMC, Illkirch, France)), Symplekin (Becton–Dickinson), MLL-C (Upstate Biotechnology), c-Myc (9E10, Santa Cruz Biotechnology), Acetyl Lysine (Cell Signaling Technology) specific antibodies associated with protein G agarose beads, were used for immunoprecipitating proteins with gentle shaking at 4 °C overnight.

Techniques: Expressing, Transfection, Standard Deviation, Western Blot, Quantitation Assay

Journal: Journal of Bacteriology

Article Title: Conditional Mutation of an Essential Putative Glycoprotease Eliminates Autolysis in Staphylococcus aureus

doi: 10.1128/jb.01806-06

Figure Lengend Snippet: FIG. 2. Triton X-100-induced autolysis of conditional gcp mutants. (A) spacp-regulated gcp mutant RN4220/Pspac-gcp; (B) gcp antisense mutant WCUH29/gcp-as. The indicated strains and the control, WCUH29/pYH4, were grown in TSB in the presence of different concentrations of IPTG (1 mM) or ATc (0.5 g/ml). Results were normalized to the OD580 at time zero (OD0). The percent lysis was determined as follows: percent lysis at time t [(OD0 ODt)/OD0] 100. The experiments were repeated at least three times. Each figure represents the results of one experiment.

Article Snippet: The resulting plasmid, pLZ106, was purified and electroporated into S. aureus WCUH29, resulting in strain WCUH29/pLZ106. lux expression was monitored with a Chiron luminometer.

Techniques: Mutagenesis, Control, Lysis

Journal: Journal of Bacteriology

Article Title: Conditional Mutation of an Essential Putative Glycoprotease Eliminates Autolysis in Staphylococcus aureus

doi: 10.1128/jb.01806-06

Figure Lengend Snippet: FIG. 3. Effects of penicillin G on the growth of conditional gcp mutants. (A) spacp-regulated gcp mutant RN4220/Pspac-gcp; (B) gcp antisense mutant WCUH29/gcp-as. The indicated strains and the con- trol, WCUH29/pYH4, were grown in TSB in the presence of different concentrations of inducer (either IPTG or ATc). Penicillin (20 MIC) was added to the exponential-phase cultures at a final concentration of 8 g/ml. The bacterial cultures were continuously incubated, and the OD600 values for the cultures were measured every hour for 8 h. FIG. 4. Effect of down-regulation of gcp expression on hydrolase activity. The spacp-regulated gcp mutant was grown in TSB, with or without the inducer IPTG (1 mM), and the bacterial cells were heat killed, collected by centrifugation, and washed with H2O. (A) The heat-killed cells were resuspended with fresh filter-sterilized superna- tants of overnight cultures of RN4220. The optical density of resus- pended dead cells was adjusted to 0.5 at 600 nm, and the dead cells were incubated at 37°C with shaking. The OD600 was measured every 30 min. (B and C) Zymogram analysis of the spacp-regulated gcp mutant. Equal amounts (10 g) of proteins prepared from the super- natants of different cultures of S. aureus strains were loaded and separated in 10% SDS-PAGE gels containing 0.2% heat-killed cells harvested from culture with 1 mM IPTG (B) or without IPTG (C). Lytic bands appeared as dark zones after scanning. M, protein molec- ular size marker.

Article Snippet: The resulting plasmid, pLZ106, was purified and electroporated into S. aureus WCUH29, resulting in strain WCUH29/pLZ106. lux expression was monitored with a Chiron luminometer.

Techniques: Mutagenesis, Concentration Assay, Incubation, Expressing, Activity Assay, Centrifugation, SDS Page, Marker

Journal: Journal of Bacteriology

Article Title: Conditional Mutation of an Essential Putative Glycoprotease Eliminates Autolysis in Staphylococcus aureus

doi: 10.1128/jb.01806-06

Figure Lengend Snippet: FIG. 6. Expression of lux driven by the cidA promoter in the gcp antisense expression strain WCUH29/gcp-as. Promoter activation was represented as the mean light intensity/OD600 ratio from triplicate readings at different times during growth. RLU, relative light units.

Article Snippet: The resulting plasmid, pLZ106, was purified and electroporated into S. aureus WCUH29, resulting in strain WCUH29/pLZ106. lux expression was monitored with a Chiron luminometer.

Techniques: Expressing, Activation Assay