Journal: Current cancer drug targets

Article Title: Targeting Epigenetics through Histone Deacetylase Inhibitors in Acute Lymphoblastic Leukemia

doi:

Figure Lengend Snippet: Non-Histone Proteins Known to be Direct Substrates of HDAC Enzymes

Article Snippet: α-Tubulin , Cytoskeleton , [ 69 ].

Techniques: Binding Assay, Transduction, Activation Assay

Journal: PLoS Pathogens

Article Title: A Bacterial Acetyltransferase Destroys Plant Microtubule Networks and Blocks Secretion

doi: 10.1371/journal.ppat.1002523

Figure Lengend Snippet: ( A ) HIS-HopZ1a, HIS-HopZ1a(C216A) and GST were immobilized on the surface of a Biacore CM5 sensor chip at the following response units (RU), 9953 RU, 8784 RU and 3800 RU, respectively. 500 µg/ml of bovine brain tubulin was flowed across the recombinant HopZ1a, HopZ1a(C216A) and GST -bound surface, generating a RU difference of 1049 RU, 1150 RU and −12.2 RU, respectively. The start and end of tubulin injection are indicated by arrows. ( B ) 6973 RU, 13598 RU or 18289 RU of HIS-HopZ1a was immobilized on a Biacore CM5 sensor chip. 500 µg/ml of bovine brain tubulin was flowed across the recombinant HopZ1a-bound surfaces, generating a RU difference of 59 RU, 324 RU and 615 RU, respectively. ( C ) Immunoblot analysis of HIS-HopZ1a and HIS-HopZ1a(C216A) proteins in a microtubule co-sedimentation assay detected with rabbit α-HIS antibody. In the absence of microtubules, HIS-HopZ1a and HIS-HopZ1a(C216A) proteins were found only in the supernatant (S) fractions. In the presence of microtubules, HIS-HopZ1a and HIS-HopZ1a(C216A) proteins were found predominantly in the pellet (P) fractions. ( D ) HopZ1a binds to tubulin in planta . 1.5 ml of taxol-treated clarified extracts from transgenic Arabidopsis expressing HA-tagged HopZ1a(C216A) or HopF2 were bound to 30 µl of α-HA antibody-coated agarose beads. Proteins were eluted from the α-HA beads by boiling the beads in 100 µl of Laemmli sample buffer. 5 µl, 2.5 µl and 1.25 µl of the resulting eluates (corresponding to 5%, 2.5% and 1.25% of eluates) were subjected to immunoblots using α-HA antibodies (top panel) or α-tubulin antibodies (bottom panel). In the top panel 20 ul of HopZ1a clarified extract and 5 ul of HopF2 clarified extract was loaded whereas in the bottom panel 3 ul of each clarified extract was loaded, The band intensities of HopZ1a, HopF2, and tubulin bands were quantified by ImageJ and shown at the bottom of each panel. The maximum band intensity observed in each blot is arbitrarily set at 1 and all other band intensities are shown relative to that value. The ratios of tubulin:HopZ1a(C216A) and tubulin:HopF2 are indicated at the bottom of the figure. (*) indicates non-specific, cross-reactive bands.

Article Snippet: 1, 2, 5, 10 or 20 µg of purified bovin e brain tubulin (Cytoskeleton Inc., USA) or 100 nM of phytic acid (Sigma #P5681, USA) was mixed with 2 µl of 14 C-acetyl CoA (56 µCi/µM) in the absence or presence of 1 µg of HopZ1a, HopZ1a(C216A), or HopZ1a(K289R) in a 20 µl reaction containing 50 mM HEPES (pH 8.0), 10% glycerol, 1 mM DTT and was incubated for 1 hour at 30°C .

Techniques: Recombinant, Injection, Western Blot, Sedimentation, Transgenic Assay, Expressing

Journal: PLoS Pathogens

Article Title: A Bacterial Acetyltransferase Destroys Plant Microtubule Networks and Blocks Secretion

doi: 10.1371/journal.ppat.1002523

Figure Lengend Snippet: Purified HIS-HopZ1a (∼42 kDa), GST-HopZ1a (∼68 kDa) and HIS-HopZ1a(C216A) (∼42 kDa) proteins were incubated with or without 10 µg of tubulin heterodimers (∼55 kDa) or 100 nM phytic acid in the presence of 14 C-labeled acetyl-CoA for 1 hour at 30°C. The acetyltransferase activity of HopZ1a is activated by phytic acid. Active HopZ1a autoacetylates in cis and acetylates tubulin. All samples were separated by 12% SDS-PAGE and the 14 C-incorporation was analyzed by Phosphorimager.

Article Snippet: 1, 2, 5, 10 or 20 µg of purified bovin e brain tubulin (Cytoskeleton Inc., USA) or 100 nM of phytic acid (Sigma #P5681, USA) was mixed with 2 µl of 14 C-acetyl CoA (56 µCi/µM) in the absence or presence of 1 µg of HopZ1a, HopZ1a(C216A), or HopZ1a(K289R) in a 20 µl reaction containing 50 mM HEPES (pH 8.0), 10% glycerol, 1 mM DTT and was incubated for 1 hour at 30°C .

Techniques: Purification, Incubation, Labeling, Activity Assay, SDS Page

Journal: PLoS Pathogens

Article Title: A Bacterial Acetyltransferase Destroys Plant Microtubule Networks and Blocks Secretion

doi: 10.1371/journal.ppat.1002523

Figure Lengend Snippet: ( A ) The protein sequence of HopZ1a is aligned with HopZ1b, HopZ2 and PopP2 using Clustal W. The region flanking the conserved lysine residue is shown, with lysine 289 (in HopZ1a) indicated by a star. ( B ) Purified recombinant GST-HopZ1a, GST-HopZ1a(C216A) and GST-HopZ1a (K289R) proteins were incubated with tubulin heterodimers in the presence of 14 C-labeled acetyl-CoA for 1 hour at 30°C. All samples were separated by 12% SDS-PAGE and the 14 C-incorporation was analyzed by Phosphorimager. ( C ) Macroscopic HR of Arabidopsis Col-0 leaves infiltrated with 2×10 7 CFU/ml of Pto DC3000 expressing pUCP20-hopZ1a-HA (HopZ1a WT), pUCP20-hopZ1a(C216A)-HA [HopZ1a (C216A)] or pUCP20-hopZ1a(K289R)-HA [HopZ1a(K289R)]. (*) indicate HR. ( D ) Quantification of HR by electrolyte leakage of Arabidopsis Col-0 leaf discs after infiltration with 5×10 7 CFU/ml of Pto DC3000 expressing empty vector (EV), pUCP20-hopZ1a-HA (HopZ1a WT), pUCP20-hopZ1a(C216A)-HA [HopZ1a (C216A)], or pUCP20-hopZ1a(K289R)-HA [HopZ1a(K289R)]. Error bars represent standard error and (*) indicate statistically significant differences (2-tailed student t-test, p<0.01). The experiment was repeated twice with similar results. ( E ) P. syringae ( Pci 0788-9) growth assay in Arabidopsis. Pci 0788-9 carrying pUCP20-hopZ1a-HA (HopZ1a WT) grew significantly better than Pci 0788-9 carrying pUCP20-hopZ1a(K289R)-HA [HopZ1a(K289R)] or empty vector (EV) on day 3. The bacterial growth difference between HopZ1a WT and HopZ1a K289R or EV was statistically significant [as indicated by (*), 2-tailed student t-test, p<0.01]. Error bars represent standard error. Experiments were repeated three times and the data from one representative experiment is presented.

Article Snippet: 1, 2, 5, 10 or 20 µg of purified bovin e brain tubulin (Cytoskeleton Inc., USA) or 100 nM of phytic acid (Sigma #P5681, USA) was mixed with 2 µl of 14 C-acetyl CoA (56 µCi/µM) in the absence or presence of 1 µg of HopZ1a, HopZ1a(C216A), or HopZ1a(K289R) in a 20 µl reaction containing 50 mM HEPES (pH 8.0), 10% glycerol, 1 mM DTT and was incubated for 1 hour at 30°C .

Techniques: Sequencing, Purification, Recombinant, Incubation, Labeling, SDS Page, Expressing, Plasmid Preparation, Growth Assay

Journal: The Journal of comparative neurology

Article Title: Proteomic analyses of nucleus laminaris identified candidate targets of the fragile X mental retardation protein

doi: 10.1002/cne.24281

Figure Lengend Snippet: Primary antibodies used for immunocytochemistry and other staining. Abbreviations: ICC: immunocytochemistry; WB: Western blot.

Article Snippet: Specific secondary HRP-conjugated antibodies were used at 1:2500 dilution (Santa Cruz, Biotechonology®, Inc., Dallas, TX) and blots were developed with SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific, Inc., Waltham, MA) and exposed to X-ray film. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Immunogen Manufacturer, catalog number, Host, monoclonal or polyclonal; RRID, Concentration Anti-MAP2 Bovine brain MAP2 (aa 997–1332) Millipore (Billerica, MA), MAB3418; Mouse monoclonal; RRID:AB_94856 1:1000 (ICC); 1:1000 (WB) Anti-MAP2 Synthetic peptide rat MAP2 (aa 1-100) Abcam (Cambridge, MA), ab32454; Rabbit polyclonal; RRID:AB_776174 1:1000 (ICC) Anti-MAP1B Full length rat brain MAP1B Abcam, Ab11266; Mouse monoclonal IgG1 clone AA6; RRID:AB_297884 1:10,000 (ICC); 1:1000 (WB) Anti-TuJ-1 Rat brain tubulin beta-3 R&D Systems (Minneapolis, MN), MAB1195; Mouse monoclonal IgG2a; RRID:AB_357520 1:10,000 (ICC); 1:1000 (WB) Anti-eEF1a Crude calmodulin-binding proteins from Trypanosoma brucei chromatography Millipore, 05-235; Mouse monoclonal IgG1k clone CBP-KK1; RRID:AB_309663 1:1000 (ICC); 1:1000 (WB) Anti-p-eEF2 Synthetic phosphopeptide surrounding Thr56 of human eEF2, GETRFtDTRK Cell Signaling Technology (Danvers, MA), No. 2331; Rabbit polyclonal; RRID:AB_2277755 1:1000 (ICC) Anti-NSF-1 Recombinant human full length NSF.

Techniques: Immunocytochemistry, Staining, Western Blot, Concentration Assay, Chromatography, Recombinant, Purification

Journal: The Journal of comparative neurology

Article Title: Proteomic analyses of nucleus laminaris identified candidate targets of the fragile X mental retardation protein

doi: 10.1002/cne.24281

Figure Lengend Snippet: Subcellular distribution of cytoskeletal elements and their associated proteins in NL examined by immunocytochemistry. A: Two antibodies for MAP2 raised in rabbit (r) or mouse (m) display identical dendritic labeling in NL. These two antibodies are subsequently used as dendritic markers for examining the localization of other protein candidates. B: Double labeling of MAP1B and MAP2. Strong MAP1B immunoreactivity in NL neuropil regions does not overlap with MAP2-labeled dendrites. Detectable MAP1B immunoreactivity is found in some (white star) but not other NL cell bodies (solid white circles). C: Double labeling of TuJ-1 and MAP2. TuJ-1 immunoreactivity is strong in the cell bodies and the primary portions of dendrites, and relatively weaker in the more distal dendritic branches. D: Phalloidin stain visualizing the distribution of F-actin surrounding MAP2 labeled dendritic branches (arrowheads). Arrows point to phalloidin stain along blood vesicles. Abbreviations: NM, nucleus magnocellularis; NL, nucleus laminaris; TuJ-1, neuron-specific class III beta-tubulin; MAP2, microtubule-associated protein 2; MAP1B, microtubule-associated protein 1B. Scale bar = 100 μm (left column) and 20 μm (all other columns).

Article Snippet: Specific secondary HRP-conjugated antibodies were used at 1:2500 dilution (Santa Cruz, Biotechonology®, Inc., Dallas, TX) and blots were developed with SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Scientific, Inc., Waltham, MA) and exposed to X-ray film. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Immunogen Manufacturer, catalog number, Host, monoclonal or polyclonal; RRID, Concentration Anti-MAP2 Bovine brain MAP2 (aa 997–1332) Millipore (Billerica, MA), MAB3418; Mouse monoclonal; RRID:AB_94856 1:1000 (ICC); 1:1000 (WB) Anti-MAP2 Synthetic peptide rat MAP2 (aa 1-100) Abcam (Cambridge, MA), ab32454; Rabbit polyclonal; RRID:AB_776174 1:1000 (ICC) Anti-MAP1B Full length rat brain MAP1B Abcam, Ab11266; Mouse monoclonal IgG1 clone AA6; RRID:AB_297884 1:10,000 (ICC); 1:1000 (WB) Anti-TuJ-1 Rat brain tubulin beta-3 R&D Systems (Minneapolis, MN), MAB1195; Mouse monoclonal IgG2a; RRID:AB_357520 1:10,000 (ICC); 1:1000 (WB) Anti-eEF1a Crude calmodulin-binding proteins from Trypanosoma brucei chromatography Millipore, 05-235; Mouse monoclonal IgG1k clone CBP-KK1; RRID:AB_309663 1:1000 (ICC); 1:1000 (WB) Anti-p-eEF2 Synthetic phosphopeptide surrounding Thr56 of human eEF2, GETRFtDTRK Cell Signaling Technology (Danvers, MA), No. 2331; Rabbit polyclonal; RRID:AB_2277755 1:1000 (ICC) Anti-NSF-1 Recombinant human full length NSF.

Techniques: Immunocytochemistry, Labeling, Staining