Journal: The Journal of Pediatrics

Article Title: Birth Prevalence of Homocystinuria in Central Europe: Frequency and Pathogenicity of Mutation c.1105C>T (p.R369C) in the Cystathionine Beta-Synthase Gene

doi: 10.1016/j.jpeds.2008.09.015

Figure Lengend Snippet: Quaternary structure of p.R369C mutant under different expression conditions. The p.R369C mutant was expressed in both E. coli and CHO cell-based systems, crude extracts were electrophoresed in gradient polyacrylamide gels under non-denaturing conditions followed by Western blotting (see Methods for details). A representative gel is shown, the sharply demarcated fractions (CBS) 4 , (CBS) 8 , or (CBS) 12 contain 4, 8, or 12 CBS subunits, respectively; the smear in the high molecular weight fraction contains misfolded CBS. The individual lanes contain either the p.R369C mutant (M) , wild-type CBS (WT) , or blank (B , i.e. either E. coli transformed with empty vector or CHO cells transfected with empty vector pTRE2hyg, respectively). The line “Average amount of tetramers/oligomers” shows the relative amounts of the sum of correctly folded mutant tetramers and oligomers relative to the wild-type CBS enzyme, data are means of 3 independent expressions with SD in parentheses.

Article Snippet: For Western blot analysis, cell lysates containing total protein 10 μg were electrophoresed on 4% to 15% gradient native polyacrylamide gels (BioRad precast gels) with Laemmli buffer system with or without SDS for SDS-PAGE and native-PAGE gel, respectively.

Techniques: Mutagenesis, Expressing, Western Blot, High Molecular Weight, Transformation Assay, Plasmid Preparation, Transfection

Journal: bioRxiv

Article Title: Proteasomal activity is differentially regulated in source and sink tissues of Arabidopsis

doi: 10.1101/2022.11.16.516810

Figure Lengend Snippet: Proteasome activity declines with age but is induced by cytokinin. A, Suc-LLVY-AMC (100 μM) stain of total lysates resolved on 4% Native-PAGE showing the abundance and peptidase activity of different proteasome particles (DC: double-capped 26S proteasome; SC: single-capped 26S proteasome; 20S: 20S proteasome). Total protein from the first leaf pair was isolated at day 15 (young), day 30 (mature) and day 43 (senesced) and three independent biological replicates were loaded on gel. B, Bar graphs show relative activity for the different proteasomal complexes as based on digital quantification of band intensities from the activity gels. Data is expressed relative to the activity present at day 15, which was set at 1.0. Significant differences were determined using one-way ANOVA with post-hoc Tukey HSD Test. Different letters indicate significant differences (p < 0.05). C, Dark-induced leaf senescence was performed with first leaves isolated from 30-d-old Arabidopsis plants. Leaves were floated on water containing DMSO or 30μM MG132. Pictures show leaves before and after 5 days in darkness. After the treatment the chlorophyll content was determined. Bars indicate chlorophyll content and different letters indicate significant differences according to one-way ANOVA and post-hoc Tukey HSD Test (p < 0.05). D, Proteasomal activity gels (Suc-LLVY-AMC stained) of total lysates from cytokinin or mock treated plants, legend as in A. E, Bar graphs show relative activity for the different proteasomal complexes as based on quantification of band intensities. The activity after cytokinin treatment is expressed relative to the activity without, which was set at 1.0. Asterisks indicate significant differences, Student’s t test (p < 0.05).

Article Snippet: Detached leaves from first pair of 29 days-old wild type Arabidopsis leaves were floated in the solution (water+DMSO), 30μM MG132 (N-(benzyloxycarbonyl)-leucinyl-leucinyl-leucinal; SelleckChem), 10μM Cytokinin+DMSO, 30μM MG132 + 10μM Cytokinin solution respectively, and incubated under darkness until leaves turned yellow, followed by harvesting samples for the subsequent experiments.

Techniques: Activity Assay, Staining, Clear Native PAGE, Isolation

Journal: bioRxiv

Article Title: Proteasomal activity is differentially regulated in source and sink tissues of Arabidopsis

doi: 10.1101/2022.11.16.516810

Figure Lengend Snippet: Proteasomal gene regulation during proteotoxic stress and leaf senescence. A, Effects of proteotoxic stress on the expression of proteasome promoter:GUS transgenes. Transgenic lines were grown in ½ strength Murashige and Skoog (MS) with/without 15μM MG132 for 10 days and then incubated overnight with the X-Gluc substrate. B, Proteasomal subunit abundance during proteotoxic stress. Col-0 seedling of 8 days were treated for for 2 days with either 30μM MG132, 2μM bortezomib or DMSO as a control. Total protein was separated and blotted and individual subunits were detected using specific antibodies. Coomassie Brilliant Blue (CBB) stained gels were used loading control. C, Expression of proteasomal subunit genes during leaf senescence as visualized by promoter:GUS transgenes. Shown are the results for the first leaf pair, harvested either from 15-d-old plants (young) or 43-d-old plants (old). D, A heat map summarizing the expression of proteasomal subunit genes during leaf ageing. The expression of proteasomal genes is shown for 5 time points, whereby the expression levels is given as compared to the level at day 15 (Log2FC). E, Proteasomal subunit abundance during leaf ageing. The first leaf pair of Col-0 was harvested at the given time point, and total protein was separated and blotted. The abundance for selected subunits was detected using specific antibodies. CBB stained gels were used loading control.

Article Snippet: Detached leaves from first pair of 29 days-old wild type Arabidopsis leaves were floated in the solution (water+DMSO), 30μM MG132 (N-(benzyloxycarbonyl)-leucinyl-leucinyl-leucinal; SelleckChem), 10μM Cytokinin+DMSO, 30μM MG132 + 10μM Cytokinin solution respectively, and incubated under darkness until leaves turned yellow, followed by harvesting samples for the subsequent experiments.

Techniques: Expressing, Transgenic Assay, Incubation, Control, Staining

Journal: Nature Communications

Article Title: Two KaiABC systems control circadian oscillations in one cyanobacterium

doi: 10.1038/s41467-024-51914-5

Figure Lengend Snippet: a YTH interaction analysis between KaiA3 and KaiC3. The KaiA1 dimer interaction was used as a positive control. YTH reporter strains carrying the respective bait and prey plasmids were selected by plating on complete supplement medium (CSM) lacking leucine and tryptophan (-Leu -Trp). AD, GAL4 activation domain; BD, GAL4 DNA-binding domain; empty, bait, and prey plasmids without protein sequence (only AD/BD domain). The physical interaction between bait and prey fusion proteins was determined by growth on complete medium lacking leucine, tryptophan, and histidine (-Leu -Trp -His) and the addition of 12.5 mM 3-amino-1,2,4-triazole (3-AT). The BD was fused to the N-terminus of KaiA3. For clarity, spots were assembled from several replicate assays (the original scans are shown in Fig. ). KaiC3-KaiA3 interaction analysis was performed thrice. b Interaction analysis of recombinant Kai proteins on native polyacrylamide gel. Proteins were incubated for 16 h at 30 °C and subsequently subjected to 4–16% clear native PAGE. Gels were either stained with Coomassie blue (left side) or blotted and immunodecorated with a monoclonal anti-His antibody to detect recombinant KaiA3-His6 (right side). Representative images of three independent experiments. Recombinant Synechococcus KaiA was used for comparison. c KaiC3 phosphorylation depends on the presence of KaiA3 and KaiB3. KaiC3 was dephosphorylated by incubating for 18 h at 30 °C prior to the start of the assay. 0.2 µg/µl NP-KaiC3 was incubated at 30 °C in the presence or absence of 0.1 µg/µl Synechocystis KaiA3 (A3), KaiB3 (B3) and KaiB1 (B1) and Synechococcus KaiA (A), respectively. Aliquots were taken at 0 and 16 h, followed by separation on a high-resolution LowC SDS-PAGE gel in Tris-Tricine buffer and staining with Coomassie blue. A slow-migrating band representing the phosphorylated form of KaiC3 (P-KaiC3) was observed only in the presence of KaiA3. NP indicates dephosphorylated KaiC3. Phosphorylation analysis was performed at least three times, and a negative control for Synechococcus KaiA was performed twice. Source data are provided as a Source Data file.

Article Snippet: Kai proteins (containing 0.2 μg/μl dephosphorylated Strep-KaiC3, 0.1 μg/μl KaiA3-His6, KaiB3, KaiB1 or Synechococcus KaiA, respectively) were incubated for 16 h at 30 °C in phosphorylation assay buffer, followed by separation of the native proteins in 4–16% native PAGE at 4 °C using a clear native buffer system (Serva) without anionic dye.

Techniques: Positive Control, Activation Assay, Binding Assay, Sequencing, Recombinant, Incubation, Clear Native PAGE, Staining, Comparison, Phospho-proteomics, SDS Page, Negative Control