Journal: Molecular Cell

Article Title: Initiation of Quality Control during Poly(A) Translation Requires Site-Specific Ribosome Ubiquitination

doi: 10.1016/j.molcel.2016.11.039

Figure Lengend Snippet:

Article Snippet: GST-UBE1 (human) , Boston Biochem , Cat. #E-306.

Techniques: Recombinant, Protease Inhibitor, Methylation, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Sequencing, Negative Control, Software

Journal: Molecular Biology of the Cell

Article Title: The Rho-GEF Gef3 interacts with the septin complex and activates the GTPase Rho4 during fission yeast cytokinesis

doi: 10.1091/mbc.E14-07-1196

Figure Lengend Snippet: Gef3 binds to the Rho GTPase Rho4 in vitro. (A) Coomassie blue staining of SDS–PAGE showing purified 3FLAG-Gef3 from S. pombe . The asterisk marks the 3FLAG-Gef3 band with the expected size. (B, C) Gef3 binds to Rho4 in pull-down assays. Purified GST-Rho GTPases and GST control were bound to beads, depleted for nucleotides, and then incubated with purified 3FLAG-Gef3. The amount of pulled-down Gef3 was detected by Western blotting (B; representative of four independent assays) and quantified (C; mean ± 1 SD). The intensities of 3FLAG-Gef3 bands were normalized by the intensities of pulled-down Rho GTPases. The intensity of 3FLAG-Gef3 band in GST control was set as 1.

Article Snippet: Rho GTPases were detected by monoclonal anti-GST antibody (3G10/1B3, 1:10,000 dilution; NB600-446; Novus Biologicals, Littleton, CO).

Techniques: In Vitro, Staining, SDS Page, Purification, Control, Incubation, Western Blot

Journal: Cell Reports

Article Title: Raptor-Mediated Proteasomal Degradation of Deamidated 4E-BP2 Regulates Postnatal Neuronal Translation and NF-κB Activity

doi: 10.1016/j.celrep.2019.11.023

Figure Lengend Snippet: Increased Binding to Raptor and CUL4B Boosts 4E-BP2 Proteasomal Degradation (A–C) Top: representative immunoblots from HEK293H lysates co-transfected with myc-Raptor (A). WT HA-tagged 4E-BP2 and empty vector or myc-raptor (B) co-transfected with siRNA (scrambled or against RAPTOR) and 2D HA-tagged 4E-BP2, or (C) HA-4E-BP2 ΔTOS with empty vector or myc-Raptor + HA-4E-BP2 ΔTOS. Bottom: quantification of HA expression (corresponding to WT or 2D) measured by immunoblotting, normalized to β - actin. In (C, bottom), a depiction of 4E-BP2 domains is shown to highlight the C-terminal deletion of the TOS motif (ΔTOS). (D) Representative immunoblots from HEK293H immunoprecipitates (IP; top, with anti-myc antisera) and whole lysates (bottom), co-transfected with myc-Raptor and WT or 2D HA-tagged 4E-BP2 and probed with the antisera against the indicated proteins; β - actin is the loading control. The red arrow shows increased expression in IP. Right: depiction of the Raptor-CUL4B-DDB1-2D complex. (E) Quantification of data in (D) for HA expression and CUL4B bound to myc-Raptor, Student’s t test (n = 2), ∗ p < 0.05. (F) In vitro ubiquitination assay of purified GST-4E-BP2 WT or 2D. The reactions were performed in the presence of purified CUL4B, Raptor, DDB1, His-ubiquitin, UBE2L3, and UBE1 proteins and probed with the antisera against the indicated proteins. GST, glutathione S-transferase. (G) Quantification of ubiquitination data in (E); Student’s t test (n = 3); ∗ p < 0.05. For (A)–(C), all of the experiments were carried out in the presence of 100 μg/mL cycloheximide (CHX) for 0, 1, or 2 h; β - actin is the loading control. The data are shown as means ± SEMs (error bars); n = 3 per condition; two-way ANOVA; Bonferroni’s post hoc; ∗∗ p < 0.01, ∗∗ p < 0.001. See also Figure S5 and .

Article Snippet: In vitro ubiquitination assay was performed in 100 μL reaction mixture at 37°C for 2 h. The reaction mixture included 100 ng purified human recombinant 4E-BP2 WT and N99D/N102D, 100 ng purified human recombinant UBE1 (E1 enzyme, E-304, BostonBiochem), 500 ng UbcH7/UBE2L3 (E2 enzyme, E2-640, BostonBiochem), 10 μg ubiquitin (U-530, BostonBiochem), 2.5 μg purified human recombinant CUL4B (E3 enzyme, H00008450-P01, Novus Biologicals), 50 ng purified human recombinant DDB1 (ab114333, abcam), purified human recombinant Raptor (H00057521-P01, Novus Biologicals) in an ATP-regenerating system [50 mM Tris-HCl, pH 7.6, 10 mM MgCl 2 , 2 mM ATP (R0441, ThermoFisher Scientific) 10 mM creatine phosphate (10621714001, Merck), 3.5 U/mL creatine kinase (10127566001, Merck) and 0.6 U/mL inorganic pyrophosphatase (M0361S, New England Biolabs)], in the presence of 5 μM ubiquitin aldehyde (U-201, BostonBiochem) and 50 μM MG132.

Techniques: Binding Assay, Western Blot, Transfection, Plasmid Preparation, Expressing, Control, In Vitro, Ubiquitin Proteomics, Purification

Journal: Cell Reports

Article Title: Raptor-Mediated Proteasomal Degradation of Deamidated 4E-BP2 Regulates Postnatal Neuronal Translation and NF-κB Activity

doi: 10.1016/j.celrep.2019.11.023

Figure Lengend Snippet:

Article Snippet: In vitro ubiquitination assay was performed in 100 μL reaction mixture at 37°C for 2 h. The reaction mixture included 100 ng purified human recombinant 4E-BP2 WT and N99D/N102D, 100 ng purified human recombinant UBE1 (E1 enzyme, E-304, BostonBiochem), 500 ng UbcH7/UBE2L3 (E2 enzyme, E2-640, BostonBiochem), 10 μg ubiquitin (U-530, BostonBiochem), 2.5 μg purified human recombinant CUL4B (E3 enzyme, H00008450-P01, Novus Biologicals), 50 ng purified human recombinant DDB1 (ab114333, abcam), purified human recombinant Raptor (H00057521-P01, Novus Biologicals) in an ATP-regenerating system [50 mM Tris-HCl, pH 7.6, 10 mM MgCl 2 , 2 mM ATP (R0441, ThermoFisher Scientific) 10 mM creatine phosphate (10621714001, Merck), 3.5 U/mL creatine kinase (10127566001, Merck) and 0.6 U/mL inorganic pyrophosphatase (M0361S, New England Biolabs)], in the presence of 5 μM ubiquitin aldehyde (U-201, BostonBiochem) and 50 μM MG132.

Techniques: Produced, Ubiquitin Proteomics, Reporter Assay, Western Blot, RNA Sequencing, Recombinant, Plasmid Preparation, Software, Imaging

Journal: PLoS ONE

Article Title: Plasmodium falciparum FIKK Kinase Members Target Distinct Components of the Erythrocyte Membrane

doi: 10.1371/journal.pone.0011747

Figure Lengend Snippet: A. The adhesion phenotypes of the two KO parasite lines were investigated. The FIKK7.1- and FIKK12-KO parasites and wild type FCR3 were selected on cells expressing either CSA (white bar) or CD36 (black bar), after 3 rounds of selection all the three parasite lines showed similar binding densities on BeWo (CSA) or CHO745-CD36 cells. B. Cellular localization of A-type Rifin, Stevor and Surfin 4.2 in FIKK-KO and wild type parasites. Antigen was detected using specific anti-Rifin, Surfin or Stevor antibodies by immunofluorescence assay.

Article Snippet: Briefly, plastic Petri dishes were coated overnight at 4°C with phosphate-buffered saline (PBS) containing 1 mg/ml CSA sodium salt from bovine trachea (Sigma), 1 mg/ml chondroitin sulfate C sodium salt from shark cartilage (Sigma), 10 μg/ml recombinant human CD36 (R&D Systems).

Techniques: Expressing, Selection, Binding Assay, Immunofluorescence