Journal: Molecular Plant Pathology

Article Title: Nitrate‐inducible MdBT2 acts as a restriction factor to limit apple necrotic mosaic virus genome replication in Malus domestica

doi: 10.1111/mpp.13166

Figure Lengend Snippet: MdBT2 promotes the ubiquitination and degradation of 1a in vivo and in vitro. 1a protein degradation in total proteins extracted from different transgenic apple calli in the absence (a) or presence (b) of the proteasome inhibitor MG132 in a cell‐free protein degradation assay. 1a‐HIS protein was incubated with protein extracts from transgenic ( MdBT2‐ OE [overexpression] and MdBT2‐ anti [antisense]) and wild‐type (WT) apple calli for the indicated times. Anti‐HIS was used to detect the target proteins. MdACTIN served as a loading control. The charts on the right side show the protein degradation trends in (a) (upper chart) and (b) (lower chart). The ordinate shows the ratio of band intensity at each time point compared to that at 0 h and the intensity of the protein bands at 0 h was set to 1.0. The 35S :: 1a‐HA construct was transiently expressed in WT, MdBT‐ OE, and MdBT2‐ anti apple (GL3) leaves, and the leaves were collected for protein (c) and RNA (d) extraction 5 days after agro‐infiltration. Anti‐HA antibody was used to detect the 1a‐HA protein in a western blot assay (c). MdACTIN served as a loading control in the western assay. (d) Reverse transcription quantitative PCR was used to measure the transcript level of 1a in 35S :: 1a‐HA transiently expressed in WT, MdBT‐ OE, and MdBT2‐ anti apple leaves. Bars indicate mean values of relative transcript levels of 1a. The mean values were calculated from six repeats, for which the whole experiments were repeated twice with three technical repeats in each time. Error lines with the bars indicate SD of the six repeats. Relative 1a transcript level of WT was set to 1.0. (e) MdBT2‐mediated 1a protein ubiquitination in vitro. Active MdBT2‐GFP protein was immunoprecipitated from 35S :: MdBT2‐GFP transgenic apple callus using anti‐GFP antibody and incubated with 1a‐HIS protein, E1, E2, and ubiquitin in vitro. GFP (green fluorescent protein) immunoprecipitated from 35S :: GFP transgenic apple calli served as a control. Anti‐HIS (upper panel) and anti‐Ubi (lower panel) antibodies were used to detect the target proteins. Anti‐GFP antibody was used to detect the input of GFP and the active MdBT2‐GFP proteins. 1a‐HIS and Ubi(n)‐1a‐HIS are labelled on the left side. (f) MdBT2‐mediated 1a‐HA protein ubiquitination in vivo. A 35S :: 1a ‐ HA construct was transiently expressed in WT, MdBT2‐ OE, and MdBT2‐ anti apple leaves, and the samples were collected for protein extraction 4 days after infiltration. Anti‐HA antibody was used for immunoprecipitation (IP), and anti‐HA (upper panel) and anti‐Ubi (lower panel) antibodies were used for immunoblotting (IB). Input indicates the samples collected before IP and detected with anti‐Actin antibody. 1a‐HA and Ubi(n)‐1a‐HA are labelled on the left side. Representative images of three independent experiments are shown here

Article Snippet: The ApNMV 1a‐HIS protein obtained from the prokaryotic expression system was incubated with or without MdBT2‐GFP active proteins in incubation buffer at 30°C for 12 h. The incubation buffer consisted of 50 mM Tris (pH 7.5), 2 mM dithiothreitol (DTT), 50 mM MgCl 2 , 2 mM ATP, 100 ng human E1 (recombinant human His6 UBE1; BostonBiochem), 100 ng human E2 (recombinant human UbcH5b/UBE2D2, BostonBiochem), and 1 μg ubi (recombinant human Myc Ubiquitin; BostonBiochem).

Techniques: In Vivo, In Vitro, Transgenic Assay, Degradation Assay, Incubation, Over Expression, Construct, Western Blot, Real-time Polymerase Chain Reaction, Immunoprecipitation, Protein Extraction

Journal: eLife

Article Title: A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation

doi: 10.7554/eLife.08536

Figure Lengend Snippet: ( A ) In vitro ubiquitination of TBP by HeLa cell lysates. In vitro transcribed and translated HA-TBP was incubated with indicated HeLa cellular fractions for ubiquitination assays. After the reaction, immunopurified HA-TBP was analyzed western blot using an antibody against ubiquitin. ( B ) Fractions eluted from the ion exchange (D52) column were assayed in the presence of E1 (UBE1), E2 (UbcH5b), ubiquitin, ubiquitin aldehyde (deubiquitinase inhibitor) and MG132 (proteasome inhibitor). TBP ubiquitination was analyzed by western blots using anti-TBP antibody. ( C ) Chromatography scheme of purification of TBP E3 ligase. Hela cytoplasmic fraction (S100) was subjected to a series of chromatographic fractionation as indicated. ( D ) TBP E3 ligase migrates at a size around 440–660 kDa. Input (In) and Superose 6 fractions were assayed as in ( B ). Motilities of the peak activity (440–660 kDa) are shown (bottom). ( E ) TBP E3 ligase activity after the final Mono Q chromatography step. Reactions containing Input (In) and Mono Q fractions were performed as in ( B ). ( F ) The abundance of Huwe1 protein positively correlates with levels of TBP E3 ligase activity within selected MonoQ fractions. Protein compositions of selected MonoQ fractions were determined by Mudpit (Multidimensional Protein Identification Technology). Shown are numbers of detected peptides and percentage of coverage for Huwe1 protein in indicated fractions. DOI: http://dx.doi.org/10.7554/eLife.08536.005

Article Snippet: 2 μg of ubiquitin (U-100, Boston Biochem, Cambridge, MA), 50 ng E1 (E-305, Boston Biochem), 100 ng UbcH5b (E2-622, Boston Biochem), 2 μM ubiquitin aldehyde (U-201, Boston Biochem), 10 μM MG132 (474790, EMD Millipore) and 10 μg of S100, partial purified fractions or purified recombinant Huwe1 protein for one and a half hour.

Techniques: In Vitro, Ubiquitin Proteomics, Incubation, Western Blot, Chromatography, Purification, Fractionation, Activity Assay

Journal: eLife

Article Title: A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation

doi: 10.7554/eLife.08536

Figure Lengend Snippet: ( A ) PageBlue SDS-PAGE Gel of purified recombinant wild-type (WT), catalytic domain truncation (ΔC) and catalytic site C4341S mutant (C → S) Huwe1 proteins are shown. HiMark pre-stained protein marker (Invitrogen, highest molecular weight 460 kDa) was used to ensure the purification of full-length proteins. ( B ) Huwe1 ubiquitinates TBP in a dose-dependent manner. Titrations (twofold concentration range) of recombinant His-tag Huwe1 protein were incubated with GST-TBP supplemented with E1, UbcH5b, methyl-ubiquitin, ATP regenerating mix, and ubiquitin aldehyde. After the reaction, TBP ubiquitination was analyzed through western blot using an anti-TBP antibody. ( C ) Hect domain of Huwe1 is required for the TBP ubiquitination activity. Wildtype (WT), Hect domain truncated (ΔC) and catalytic site C4341S (C → S) Huwe1 mutants were used in the ubiquitination assay as in ( B ). ( D ) Hect domain alone is insufficient for Huwe1's TBP ubiquitination activity. Two different doses of His-tag Huwe1 (over twofold range) and Hect-domain containing E3 E6AP (over twofold range) were used in the ubiquitination assay. ( E ) Huwe1 E3 activity can be activated by UbcH5 family E2s and UbcH7. A panel of different E2 conjugating enzymes were tested by the in vitro ubiquitination assays. DOI: http://dx.doi.org/10.7554/eLife.08536.008

Article Snippet: 2 μg of ubiquitin (U-100, Boston Biochem, Cambridge, MA), 50 ng E1 (E-305, Boston Biochem), 100 ng UbcH5b (E2-622, Boston Biochem), 2 μM ubiquitin aldehyde (U-201, Boston Biochem), 10 μM MG132 (474790, EMD Millipore) and 10 μg of S100, partial purified fractions or purified recombinant Huwe1 protein for one and a half hour.

Techniques: SDS Page, Purification, Recombinant, Mutagenesis, Staining, Marker, Molecular Weight, Concentration Assay, Incubation, Ubiquitin Proteomics, Western Blot, Activity Assay, In Vitro

Journal: eLife

Article Title: A specific E3 ligase/deubiquitinase pair modulates TBP protein levels during muscle differentiation

doi: 10.7554/eLife.08536

Figure Lengend Snippet: ( A ) Recombinant Huwe1 protein ubiquitinates TBP in vitro. Titrations (twofold concentration range) of recombinant His-tag Huwe1 protein are incubated with GST-TBP supplemented with E1, UbcH5b, wild-type ubiquitin and ATP regenerating mix, ubiquitin aldehyde in in vitro ubiquitination assays. After the reaction, TBP ubiquitination was analyzed through western blot using the anti-TBP antibody. ( B ) Hect domain of Huwe1 is required for its TBP ubiquitination activity. Wildtype (WT), Hect domain truncation (ΔC) and catalytic site mutant (C → S) Huwe1 were assayed as in ( A ) to test their activities to ubiquitinate TBP. ( C ) Hect domain alone is insufficient for Huwe1's TBP ubiquitination activity. Two different doses of His-tag Huwe1 (twofold range) and recombinant E6AP protein (twofold range) are used in the ubiquitination assay as in ( A ). ( D ) Huwe1 E3 activity can be supported by UbcH5 family E2s and UbcH7. A panel of different E2 conjugating enzymes is used in the in vitro ubiquitination assays. ( E ) Huwe1 mediated the K48-linked ubiquitination of TBP. Wild-type ubiquitin (WT), lysine 11 to arginine mutant (K11R), lysine48 to arginine mutant (K48R), lysine63 to arginine mutant (K63R) and lysine-methylated ubiquitin (Methyl-Ub) are used in the ubiquitination assays. DOI: http://dx.doi.org/10.7554/eLife.08536.007

Article Snippet: 2 μg of ubiquitin (U-100, Boston Biochem, Cambridge, MA), 50 ng E1 (E-305, Boston Biochem), 100 ng UbcH5b (E2-622, Boston Biochem), 2 μM ubiquitin aldehyde (U-201, Boston Biochem), 10 μM MG132 (474790, EMD Millipore) and 10 μg of S100, partial purified fractions or purified recombinant Huwe1 protein for one and a half hour.

Techniques: Recombinant, In Vitro, Concentration Assay, Incubation, Ubiquitin Proteomics, Western Blot, Activity Assay, Mutagenesis, Methylation