Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 1. Individuals with biallelic CDK4 variants display microcephaly and short stature. (A) Family pedigrees with segregation of CDK4 variants. (Square) Male, (circle) female, (filled symbols) individuals with microcephaly, (strikethrough) deceased. WT Reference (+), variants v1 and v2, and zygosity are indicated for each studied individual. (B) Diagram of CDK4 transcript (top) and protein (bottom); coding exons are depicted as black rectangles. Red lines indicate variant location. (SS) Splice site disrupted. (C) Altered splicing predictions for the c.218G > A substitution generated using Alamut. (Blue rectangles) Strength of splice donor predictions for individual splice algo- rithms, (blue triangle) predicted donor splice site. (D) Growth parameters at birth and at last assessment (postnatal). (W) Weight, (OFC) orbito–frontal circumference. Z-scores show standard deviations from population mean for age and sex. Dashed lines indicate a 95% con- fidence interval for the general population. Individual subject data points from families A (circles) and B (squares) are graphed, and mean values are plotted. (E) MRI scan of age-matched control (4 years 8 months) and affected individuals with a CDK4 variant. Coronal FLAIR projection shows simplified parietal and temporal gyri, reduced white matter volume, and the absence of brain malformations. Scale bars, 10 cm. (See also Supplemental Figure S1C for additional MRI projections.) (F) Photographs of all affected individuals.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Variant Assay, Generated, Control

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 3. Full-length CDK4 protein is undetectable in patient fibroblasts. (A,B) Immunoblots of total cell extracts obtained from expo- nentially growing control (C1 and C2) and patient (P1 and P2) fibroblasts without (A) and with (B) CDK4 complementation. α-Tubulin was used as the loading control. A rabbit monoclonal antibody to C-terminal CDK4 was used; a different mouse CDK4 antibody raised against full-length CDK4 was used in Figure 5A. A smaller ∼12 kDa molecular weight band was variably detected in P1 with this antibody (Sup- plemental Fig. S2D) that might correspond to the 46 amino acid truncated nonfunctional protein predicted from RNA studies. (C) CDK6 and Cyclin D1 levels were unchanged in patient fibroblasts compared with wild-type controls.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Western Blot, Control, Molecular Weight

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 4. CDK4 mutations do not alter mitosis. (A) Percentage of mitotic cells (p-Histone H3 ser10-positive) in control (C1 and C2) and patient (P1 and P2) fibroblasts as measured by flow cy- tometry. Data points are from three independent experiments (two for C1); one-way ANOVA with Tukey post test; mean ± SEM. (B) Quantification of metaphase cells with more than two centrosomes, expressed as percentage. Numbers of cells analyzed were as follows: C1, 79; C2, 94; P1, 150; and P2, 101. Two-tailed t- test; mean ± SEM; measurements were pooled from two indepen- dent experiments. (C) Representative confocal images of control (C1 and C2) and patient (P1 and P2) fibroblasts fixed and stained for DAPI (gray), α-tubulin (green), and pericentrin (magenta). Scale bars, 5 µm.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Control, Two Tailed Test, Staining

Journal: Genes & development

Article Title: CDK4 loss-of-function mutations cause microcephaly and short stature.

doi: 10.1101/gad.352311.124

Figure Lengend Snippet: Figure 5. CDK4 mutations impair G1-to-S progression and lead to reduced cell proliferation. (A) Western blot of control and patient-de- rived fibroblasts with and without WT CDK4 complementation. (B, left) Growth curves of control and patient-derived fibroblasts with and without WT CDK4 complementation. (Right) Bar graph showing quantification of doubling times; one-way ANOVA with Tukey post test. P-values are indicated; mean ± SEM. (C) Cell cycle distribution (G0/G1, S, and G2/M) derived from BrdU and DNA (DAPI) flow cytometry scatter plots show fewer cells in S phase (BrdU+) in patient-derived fibroblasts compared with controls. n = 3 independent experiments; mean ± SEM. Gates are shown on representative plots at the right. (D) Cell cycle distribution after complementation of patient-derived fibroblasts with CDK4. Reduced G0/G1 and increased S-phase populations consistent with rescue of a G1/S progression defect. n = 3 in- dependent experiments; mean ± SEM. (See also Supplemental Fig. S4A.) (E) Quantification of DNA synthesis rate (BrdU mean fluorescence intensity [MFI] of gated population in the red rectangle) from experiments depicted in C.

Article Snippet: Patient fibroblasts were transduced with lentiviral particles containing pLIX_403-CDK4 and/or pLIX_403CDK6, a construct where full-length codon-optimized CDK4 or CDK6 was gateway-cloned into pLIX_403 (Addgene 41395 and 158560).

Techniques: Western Blot, Control, Derivative Assay, Flow Cytometry, DNA Synthesis, Fluorescence

Journal: Nature Communications

Article Title: Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer

doi: 10.1038/s41467-020-17647-x

Figure Lengend Snippet: a Domain layout of RNF4 to scale (residues 1–194). b Overlay of 1 H– 15 N HSQC spectra for RNF4 (32–194; yellow) and RNF4N (32–133; blue) showing near identical chemical shift patterns for the N-terminal region. Note the RNF4 spectrum was recorded with TROSY magnetisation selection and adjusted for the peak offsets. Peaks only observed in RNF4 spectrum arise from the structured C-terminal RING Domain. Near identical peak patterns for the N-terminal regions indicate that the conformational properties are the same in both samples. c Enlarged spectra corresponding to boxed area in B highlighting residues in the SIMs and SIMs/RING domain linker region.

Article Snippet: Full-length RNF4 variants (genes synthesized by GenScript) were expressed from the pLOU3 vector in Escherichia coli BL21 (DE3) cells, while RNFN 27-118 variants (gene fragments produced as gBlocks by IDT and recombined into pHis-TEV-30a using New England BioLabs HiFi assembly) were expressed from pHis-TEV-30a vector in E. coli Rosetta (DE3) cells as previously described , .

Techniques: Selection

Journal: Nature Communications

Article Title: Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer

doi: 10.1038/s41467-020-17647-x

Figure Lengend Snippet: a NMR structure (PDB: 2MP2) of the SIM2/3 region of RNF4 (orange) bound to a SUMO2 dimer (cyan) modified to include the donor (green) and acceptor (red) fluorophores used for single-molecule analysis of RNF4, with a predicted FRET efficiency (E FRET ) of 0.6 (left). A modelled peptide (PRB: 5M1U) with similar interdye distance but a stretched conformation produced a predicted E FRET value of 0.23 (right). b Normalised single-molecule FRET histograms of RNF4N peptides with positions of the donor (green) and acceptor (red) dyes indicated (labelled residue numbers inset). RNF4N was measured in the absence (blue) and presence (purple) of SUMO chains. Arrows indicate FRET populations used for comparison with/without SUMO (in c ). Single-molecule histograms were built from more than 500 molecules. c Relative change in E FRET upon SUMO addition (dark yellow) from b was calculated as: E FRET (+SUMO) − E FRET (−SUMO). d Representative single-molecule trajectories obtained for RNF4N peptides in b . Top panels show donor (green) and acceptor (red) intensity signals. Bottom panels show the E FRET trajectory derived from the donor/acceptor intensity traces in the absence (blue) and presence (purple) of SUMO chains. e Model of the SIM region of RNF4 displaying a compact shape in-line with the single-molecule analysis. The position of FRET dye pairs for RNF4N peptides along with E FRET values corresponding to histograms in b are shown (30/57, red; 44/70, green; 57/84, blue). Source data are provided as a Source Data file.

Article Snippet: Full-length RNF4 variants (genes synthesized by GenScript) were expressed from the pLOU3 vector in Escherichia coli BL21 (DE3) cells, while RNFN 27-118 variants (gene fragments produced as gBlocks by IDT and recombined into pHis-TEV-30a using New England BioLabs HiFi assembly) were expressed from pHis-TEV-30a vector in E. coli Rosetta (DE3) cells as previously described , .

Techniques: Modification, Produced, Residue, Comparison, Derivative Assay

Journal: Nature Communications

Article Title: Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer

doi: 10.1038/s41467-020-17647-x

Figure Lengend Snippet: a hydropathy plot of RNF4 revealing the charge characteristics of the amino-acid side chains. The SIMs and RING domain are highlighted, along with the charged linker that connects these two domains. b layout of the RNF4N peptide used for single-molecule analysis. Variants of the RNF4N peptide were produced removing either the basic or acid charge from the SIMs/RING-domain linker region. The mutations used are displayed against the WT peptide. c size exclusion chromatography profile of the WT RNF4N peptide (black) along with ∆Basic (blue) and ∆Acidic (magenta) variants. d single-molecule FRET histograms obtained from RNF4N peptides labelled with donor/acceptor FRET dyes at C-terminus and basic region (labelled residue numbers inset). Arrows inset highlight the FRET efficiency average for WT (black), ∆Basic (blue) and ∆Acidic (magenta) RNF4N peptides. Single-molecule histograms were built from more than 400 molecules. e Distance measurements over time for five independent simulations between acidic residues in the SIM2/3 linker (defined by E59) and the midpoint of the arginine-rich motif for RNF4N WT (top) and ∆Basic (bottom). f Conformational clustering of RNF4N peptides (WT top; ∆Basic bottom) generated from residue-residue contacts over the final 100 ns of each run trajectory from e . The SIMs (orange), arginine-rich region (blue) and acidic region in the SIMs/RING-domain linker (red) are highlighted. Source data are provided as a Source Data file.

Article Snippet: Full-length RNF4 variants (genes synthesized by GenScript) were expressed from the pLOU3 vector in Escherichia coli BL21 (DE3) cells, while RNFN 27-118 variants (gene fragments produced as gBlocks by IDT and recombined into pHis-TEV-30a using New England BioLabs HiFi assembly) were expressed from pHis-TEV-30a vector in E. coli Rosetta (DE3) cells as previously described , .

Techniques: Produced, Size-exclusion Chromatography, Residue, Generated

Journal: Nature Communications

Article Title: Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer

doi: 10.1038/s41467-020-17647-x

Figure Lengend Snippet: a Model of full-length RNF4 displaying potential low-FRET and high-FRET states. b Single-molecule FRET histograms obtained from full-length RNF4 WT, labelled with donor/acceptor FRET dyes at SIMs and RING domain (residues 70,144; illustrated inset) (left). Pie charts indicate the percentage of molecules in a low-FRET (green) or high-FRET (red) state. Representative single-molecule trajectories obtained for RNF4 WT (right). c As in b but analysis performed on RNF4 ∆Basic. Single-molecule histograms were built from more than 500 molecules. Source data are provided as a Source Data file.

Article Snippet: Full-length RNF4 variants (genes synthesized by GenScript) were expressed from the pLOU3 vector in Escherichia coli BL21 (DE3) cells, while RNFN 27-118 variants (gene fragments produced as gBlocks by IDT and recombined into pHis-TEV-30a using New England BioLabs HiFi assembly) were expressed from pHis-TEV-30a vector in E. coli Rosetta (DE3) cells as previously described , .

Techniques:

Journal: Nature Communications

Article Title: Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer

doi: 10.1038/s41467-020-17647-x

Figure Lengend Snippet: a Ubiquitination activity of RNF4 wild-type (WT) and ∆Basic mutant in response to increasing substrate (4xSUMO) concentrations. Assays (15 min) contained E1 activating enzyme, UbcH5a and fluorescently labelled ubiquitin. T = 0 is without ATP. Reaction products were fractionated by SDS-PAGE, followed by in-gel fluorescence. b Quantitation of fluorescent free ubiquitin in A (WT red, ∆Basic blue). Ubiquitin levels at T = 0 represented 100%. c Fluorescence polarization real-time ubiquitination assay comparing activity of WT (red) against ∆Basic (blue). Assays contained 4×SUMO, E1, UbcH5a and fluorescent ubiquitin. Reactions initiated with ATP addition and FP measured over 50 minutes. d Substrate ubiquitination activity assay containing fluorescently labelled 4×SUMO, ubiquitin, E1, UbcH5a and either RNF4 WT or RNF4 ∆Basic. Reaction products analysed as in a . e Quantitation of D for RNF4 WT (red) and RNF4 ∆Basic (blue). f Lysine discharge assays using fluorescently labelled ubiquitin. The discharge of ubiquitin from the E2 (E2~Ub) was analysed by SDS-PAGE over time in the presence of RNF4 WT, RNF4 ∆Basic, lysine only, or buffer only. g Measurement of fluorescence from the E2~Ub as seen in F. T = 0 represents 100% E2~Ub (WT, red; ∆Basic, blue; lysine only, grey; buffer only, dashed). h Binding affinity of RNF4 for 4xSUMO via FP. A fixed 4xSUMO concentration was titrated with RNF4 (WT, red; ∆Basic, blue) to saturating concentrations. For each graph ( b , c , e , g , h ) data represent mean ± standard deviation (the experiment was done in triplicate, n = 3). Experiments were carried out three times with similar results. Experiments in a , d and f were carried out three times with similar results. Source data are provided as a Source Data file.

Article Snippet: Full-length RNF4 variants (genes synthesized by GenScript) were expressed from the pLOU3 vector in Escherichia coli BL21 (DE3) cells, while RNFN 27-118 variants (gene fragments produced as gBlocks by IDT and recombined into pHis-TEV-30a using New England BioLabs HiFi assembly) were expressed from pHis-TEV-30a vector in E. coli Rosetta (DE3) cells as previously described , .

Techniques: Ubiquitin Proteomics, Activity Assay, Mutagenesis, SDS Page, Fluorescence, Quantitation Assay, Binding Assay, Concentration Assay, Standard Deviation

Journal: Nature Communications

Article Title: Functional 3D architecture in an intrinsically disordered E3 ligase domain facilitates ubiquitin transfer

doi: 10.1038/s41467-020-17647-x

Figure Lengend Snippet: Step 1, PML protein (grey) is modified with SUMO (blue), which recruits RNF4 (orange). RNF4 dimerises through its C-terminal RING domains, activating it, before binding an E2~Ub conjugate (light blue, green respectively). Steps 2–4, the compact but flexible N-terminus of RNF4 allows the RING-dimer to access the area around the SIM bound SUMOs. Step 5, this broad distribution of ubiquitin promotes rapid amplification of the SUMO/Ub signal.

Article Snippet: Full-length RNF4 variants (genes synthesized by GenScript) were expressed from the pLOU3 vector in Escherichia coli BL21 (DE3) cells, while RNFN 27-118 variants (gene fragments produced as gBlocks by IDT and recombined into pHis-TEV-30a using New England BioLabs HiFi assembly) were expressed from pHis-TEV-30a vector in E. coli Rosetta (DE3) cells as previously described , .

Techniques: Modification, Binding Assay, Ubiquitin Proteomics, Amplification

Journal: Cell Stress & Chaperones

Article Title: BAG3 and BAG6 differentially affect the dynamics of stress granules by targeting distinct subsets of defective polypeptides released from ribosomes

doi: 10.1007/s12192-020-01141-w

Figure Lengend Snippet: BAG6 is not recruited inside stress granules. a HeLa cells were treated with arsenite 0.5 mM for 45 min or heat shock (HS) at 43.5 °C for 1 h or MG132 20 μM for 3 h. Cells were fixed and subjected to immunofluorescence using antibodies specific for the SG protein G3BP and BAG6. Scale bars = 10 μm. b HeLa cells were treated with MG132 20 μM and OP-puro 25 μM for 3 h, where indicated HSP70 ATPase activity was blocked using 40 μM VER-155008. Cells were fixed and subjected to immunofluorescence using specific antibodies for G3BP and BAG6. OP-puro-labeled nascent chains were visualized by click chemistry with Alexa594-Azide. Scale bars = 10 μm

Article Snippet: Membranes were blocked with 3% BSA-Tris-buffered saline (TBS) with Tween 20 (0.01%) for 1 h at room temperature, then were incubated for 16 h at 4 °C with the following antibodies: puromycin (Merck), BAG6 (Abcam), home-made rabbit antibodies against BAG3 and HSPB8 (Carra et al. 2008a ), HSPA1A (HSP70) (StressMarq Biosciences), ubiquitin (Dako), and TUBA4A (Sigma-Aldrich).

Techniques: Immunofluorescence, Activity Assay, Labeling

Journal: Cell Stress & Chaperones

Article Title: BAG3 and BAG6 differentially affect the dynamics of stress granules by targeting distinct subsets of defective polypeptides released from ribosomes

doi: 10.1007/s12192-020-01141-w

Figure Lengend Snippet: BAG6 does not replace BAG3 in granulostasis and is not recruited inside stress granules in BAG3-depleted cells. a HeLa cells were exposed to MG132 20 μM for 3 or 6 h and protein extracts were prepared and subjected to immunoblotting to measure the expression levels of BAG3, BAG6, HSPB8, and HSPA1A. Quantitation of BAG6 protein levels is reported; n = 3, +/− SEM. b HeLa cells were lipofected for 72 h with siRNA non-targeting control or against BAG3. Cells were either left untreated (control) or exposed to MG132 (20 μM) alone or with VER-155008 (40 μM) for 3 h, fixed, and subjected to immunostaining with antibodies specific for BAG6 and the SG protein G3BP. Scale bars = 10 μm. c Quantitation of BAG6 mean ratio inside/outside SG ROI in cells from b. Number of SGs counted: 920 (siRNA control MG132); 1341 (siRNA control MG132 + VER); 419 (siRNA BAG3 MG132); 1619 (siRNA BAG3 MG132 + VER). d HeLa cells were transfected with cDNAs encoding for His-BAG3, His-BAG3-ΔBAG, His-BAG3-ΔB8, or empty vector. Twenty-four hours post-transfection, the NP-40 soluble fractions were subjected to Ni-NTA purification, and beads were processed for Western blotting using anti-BAG3 and anti-HSPB8 antibodies. e HeLa cells were transfected with cDNAs encoding for His-BAG3, His-BAG3-ΔBAG, or V5-HSP70. Twenty-four hours post-transfection, the NP-40 soluble fractions were subjected to Ni-NTA purification, and beads were processed for Western blotting using BAG3, HSP70, and HSPB8 specific antibodies. f HeLa cells were transfected with cDNAs encoding for His-BAG3, His-BAG3-ΔBAG, His-BAG3-ΔB8, or empty vector. Twenty-four hours post-transfection, cells were treated with puromycin (10 μg/ml) for 45 min and then the NP-40 soluble fractions were subjected to Ni-NTA purification and beads were processed for Western blotting using BAG3 and puromycin-specific antibodies. Quantification of the amount of puromycilated proteins pulled-down by His-BAG3, His-BAG3-ΔBAG, or His-BAG3-ΔB8 in three independent experiments is shown; **p ≤ 0.005, +/− SEM

Article Snippet: Membranes were blocked with 3% BSA-Tris-buffered saline (TBS) with Tween 20 (0.01%) for 1 h at room temperature, then were incubated for 16 h at 4 °C with the following antibodies: puromycin (Merck), BAG6 (Abcam), home-made rabbit antibodies against BAG3 and HSPB8 (Carra et al. 2008a ), HSPA1A (HSP70) (StressMarq Biosciences), ubiquitin (Dako), and TUBA4A (Sigma-Aldrich).

Techniques: Western Blot, Expressing, Quantitation Assay, Immunostaining, Transfection, Plasmid Preparation, Purification

Journal: Cell Stress & Chaperones

Article Title: BAG3 and BAG6 differentially affect the dynamics of stress granules by targeting distinct subsets of defective polypeptides released from ribosomes

doi: 10.1007/s12192-020-01141-w

Figure Lengend Snippet: Distinct functions of BAG3 and BAG6. Upon stress, newly synthesized proteins and DRiPs are released by disassembling polysomes. mRNAs and RBPs are compartmentalized inside stress granules. The VCP, HSPB8, and HSP70 chaperones bind to newly synthesized polyubiquitinated (Ub) proteins, including DRiPs. HSPB8 and HSP70 form a multiprotein complex with BAG3, p62/SQSTM1, and the motor protein dynein that targets DRiPs and polyubiquitinated proteins to LC3-positive autophagosomes for degradation. As such, DRiPs do not accumulate inside stress granules, which maintain their liquid-like properties (Ganassi et al. 2016). BAG6 also binds to HSP70 and polyubiquitinated proteins. However, in contrast to BAG3 (Fig. ​(Fig.4),4), BAG6 can directly bind to polyubiquitinated proteins and 26S proteasomes (as previously shown by Minami et al. 2010). BAG6 bound to HSP70 and 26S proteasomes is not involved in granulostasis (red cross). Instead, BAG6 targets proteins of the secretory pathway that have failed to insert into the ER to degradation via ERAD and ERpQC, in cooperation with VCP (see Kadowaki et al. 2015; Wang et al. 2011). BAG6 has also been implicated in the processing of antigens that are generated via the degradation of newly synthesized proteins and in the presentation of antigens at the cell surface, thereby participating in the immune response (see Bitzer et al. 2016).

Article Snippet: Membranes were blocked with 3% BSA-Tris-buffered saline (TBS) with Tween 20 (0.01%) for 1 h at room temperature, then were incubated for 16 h at 4 °C with the following antibodies: puromycin (Merck), BAG6 (Abcam), home-made rabbit antibodies against BAG3 and HSPB8 (Carra et al. 2008a ), HSPA1A (HSP70) (StressMarq Biosciences), ubiquitin (Dako), and TUBA4A (Sigma-Aldrich).

Techniques: Synthesized, Generated

Journal: PLoS ONE

Article Title: The Early-Acting Peroxin PEX19 Is Redundantly Encoded, Farnesylated, and Essential for Viability in Arabidopsis thaliana

doi: 10.1371/journal.pone.0148335

Figure Lengend Snippet: (A) Alignment of PEX19A and PEX19B from various plants (green) with the zebra fish ( Danio rerio ) and human ( Homo sapiens ) homologs (red), highlighting the carboxyl-terminal CaaM farnesylation motif (purple) and the domains implicated in PEX3 (brown) and PMP (blue) binding in human PEX19 [ – ]. Sequences were aligned using MegAlign program (DNAStar) and the Clustal W method. Residues identical in at least seven sequences are boxed in black, chemically similar residues are boxed in gray. The sites of the T-DNA insertions in the pex19a-1 and pex19b-1 are indicated by triangles above the sequences. (B) Phylogenetic tree showing relationships of proteins in panel A generated by the MegAlign program. The Arabidopsis PEX19A and PEX19B duplication is found in closely related plants, such as Arabidopsis lyrata and Capsella rubella , but not in more distantly related plants, such as Medicago or Brachypodium . (C) PEX19A and PEX19B gene diagrams showing T-DNA insertion sites with triangles, introns as lines, and exons as boxes.

Article Snippet: After transfer, membranes were rocked for 1 hour at 4°C in blocking buffer (8% non-fat dry milk [w/v], 20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20) and incubated overnight at 4°C with primary antibodies diluted in blocking buffer: 1:1,000 rabbit α-APX3 [ , ], 1:100 rabbit α-GFP (Clontech 632376), 1:100 rabbit α-PEX5 [ ], 1:800 rabbit α-PEX7 [ ], 1:500 rabbit α-PEX10 [ ], 1:10,000 rabbit α-PEX14 (Agrisera AS08 372), 1:500 or 1:2000 rabbit α-PEX19 (generated and affinity purified by Proteintech Group using a recombinant protein that included the full amino acid sequence of PEX19B), 1:2,000 rabbit α-PMDH2 [ ], 1:5,000 rabbit α-thiolase [ ], 1:50,000 or 1:100,000 mouse α-HSC70 (StressGen Bioreagents SPA-817), or 1:2,000 mouse α-mito ATP synthase (MitoScience MS507).

Techniques: Binding Assay, Generated

Journal: PLoS ONE

Article Title: The Early-Acting Peroxin PEX19 Is Redundantly Encoded, Farnesylated, and Essential for Viability in Arabidopsis thaliana

doi: 10.1371/journal.pone.0148335

Figure Lengend Snippet: (A) Proteins with a C-terminal CaaX motif (Cys-aliphatic-aliphatic-X; where X can be Ser, Met, Ala, Asn, or Cys) can be farnesylated by a protein farnesyl-transferase complex composed of PLP and ERA1, cleaved of the three carboxyl-terminal residues, and methylated (me) on the carboxyl group of the prenylated Cys residue . (B) Proteins with a C-terminal a CaaL motif (Cys-aliphatic-aliphatic-Leu) can be geranylgeranylated by protein geranylgeranyl-transferase complex composed of PLP and GGB . (C) PEX19 is farnesylated in vivo . Protein from 8-day-old light-grown seedlings was separated using 12% PAGE and processed for immunoblotting with antibodies recognizing PEX19 and HSC70 (loading control). The positions of the molecular mass markers (in kDa) are indicted at the right. The positions of unfarnesylated (u) and farnesylated (f) PEX19 are indicated at the left. An asterisk marks a protein that cross-reacts with the PEX19 antibody. (D) HA-PEX19 expression decreases farnesylation of endogenous PEX19. Protein extracted from 4-day-old light-grown seedlings was separated using 12% PAGE and processed for immunoblotting with antibodies recognizing PEX19 (top panel), the HA epitope (middle panel), and HSC70 (bottom panel; loading control). The positions of the molecular mass markers (in kDa) are indicted at the left. The positions of unfarnesylated (u), farnesylated (f), and HA-tagged PEX19 are indicated at the right. An asterisk marks a protein that cross-reacts with the PEX19 antibody.

Article Snippet: After transfer, membranes were rocked for 1 hour at 4°C in blocking buffer (8% non-fat dry milk [w/v], 20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20) and incubated overnight at 4°C with primary antibodies diluted in blocking buffer: 1:1,000 rabbit α-APX3 [ , ], 1:100 rabbit α-GFP (Clontech 632376), 1:100 rabbit α-PEX5 [ ], 1:800 rabbit α-PEX7 [ ], 1:500 rabbit α-PEX10 [ ], 1:10,000 rabbit α-PEX14 (Agrisera AS08 372), 1:500 or 1:2000 rabbit α-PEX19 (generated and affinity purified by Proteintech Group using a recombinant protein that included the full amino acid sequence of PEX19B), 1:2,000 rabbit α-PMDH2 [ ], 1:5,000 rabbit α-thiolase [ ], 1:50,000 or 1:100,000 mouse α-HSC70 (StressGen Bioreagents SPA-817), or 1:2,000 mouse α-mito ATP synthase (MitoScience MS507).

Techniques: Methylation, Residue, In Vivo, Western Blot, Control, Expressing

Journal: PLoS ONE

Article Title: The Early-Acting Peroxin PEX19 Is Redundantly Encoded, Farnesylated, and Essential for Viability in Arabidopsis thaliana

doi: 10.1371/journal.pone.0148335

Figure Lengend Snippet: 8-day-old wild-type, pex19a-1 , and pex19b-1 seedlings were separated into roots and aerial tissues (shoots); other tissues were collected from 31-day-old plants: rosette leaf (beginning to senesce), oldest cauline leaf, open flowers, and green siliques (third elongated silique from the apex). Extracts were separated using 10% PAGE and processed for immunoblotting with antibodies recognizing PEX19, PMDH, and HSC70. The positions of the molecular mass markers (in kDa) are indicted at the right. An asterisk marks a protein that cross-reacts with the PEX19 antibody.

Article Snippet: After transfer, membranes were rocked for 1 hour at 4°C in blocking buffer (8% non-fat dry milk [w/v], 20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20) and incubated overnight at 4°C with primary antibodies diluted in blocking buffer: 1:1,000 rabbit α-APX3 [ , ], 1:100 rabbit α-GFP (Clontech 632376), 1:100 rabbit α-PEX5 [ ], 1:800 rabbit α-PEX7 [ ], 1:500 rabbit α-PEX10 [ ], 1:10,000 rabbit α-PEX14 (Agrisera AS08 372), 1:500 or 1:2000 rabbit α-PEX19 (generated and affinity purified by Proteintech Group using a recombinant protein that included the full amino acid sequence of PEX19B), 1:2,000 rabbit α-PMDH2 [ ], 1:5,000 rabbit α-thiolase [ ], 1:50,000 or 1:100,000 mouse α-HSC70 (StressGen Bioreagents SPA-817), or 1:2,000 mouse α-mito ATP synthase (MitoScience MS507).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: The Early-Acting Peroxin PEX19 Is Redundantly Encoded, Farnesylated, and Essential for Viability in Arabidopsis thaliana

doi: 10.1371/journal.pone.0148335

Figure Lengend Snippet: (A) Light-grown pex19a-1 , pex19b-1 , and seedlings expressing HA-PEX19 display wild-type IBA sensitivity. The era1-2 , ggb-3 , and plp-4 prenylation mutants also are IBA sensitive. pex7-2 is an IBA-resistant control. Error bars show standard deviations of mean 8-day-old root lengths ( n ≥ 7). Different letters above bars indicate significantly different means (one-way ANOVA, P < 0.001). (B) Dark-grown pex19a-1 , pex19b-1 , and seedlings expressing HA-PEX19 display wild-type IBA sensitivity and sucrose independence. The prenylation mutants are also IBA sensitive and sucrose independent. pex7-2 is an IBA-resistant control. Error bars show standard deviations of mean 5-day-old hypocotyl lengths ( n ≥ 8). Different letters above bars indicate significantly different means (one-way ANOVA, P < 0.001). (C) pex19a-1 , pex19b-1 , prenylation mutants, and seedlings expressing HA-PEX19 display wild-type levels of several PMPs. Protein extracted from 4-day-old light-grown seedlings was separated using 10% PAGE and processed for immunoblotting. The membrane was serially probed with antibodies recognizing the indicated proteins. (D) pex19a-1 , pex19b-1 , prenylation mutants, and wild-type seedlings expressing HA-PEX19 fully process the PTS2 region of PMDH. Protein from 8-day-old light-grown seedlings was separated using 10% PAGE and processed for immunoblotting with antibodies recognizing PMDH or HSC70 (loading control). The positions of the molecular mass markers (in kDa) are indicted at the left. PMDH is synthesized as a precursor (p) with a cleavable PTS2 signal that is processed into the mature (m) protein in the peroxisome; this processing is impaired in the pex7-2 mutant. (E) The pex19b-1 mutant displays normal import of peroxisomally-targeted GFP. GFP fluorescence of cotyledon epidermal cells from 5-day-old light-grown seedlings carrying the 35S : PTS2-GFP construct was imaged using confocal microscopy.

Article Snippet: After transfer, membranes were rocked for 1 hour at 4°C in blocking buffer (8% non-fat dry milk [w/v], 20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20) and incubated overnight at 4°C with primary antibodies diluted in blocking buffer: 1:1,000 rabbit α-APX3 [ , ], 1:100 rabbit α-GFP (Clontech 632376), 1:100 rabbit α-PEX5 [ ], 1:800 rabbit α-PEX7 [ ], 1:500 rabbit α-PEX10 [ ], 1:10,000 rabbit α-PEX14 (Agrisera AS08 372), 1:500 or 1:2000 rabbit α-PEX19 (generated and affinity purified by Proteintech Group using a recombinant protein that included the full amino acid sequence of PEX19B), 1:2,000 rabbit α-PMDH2 [ ], 1:5,000 rabbit α-thiolase [ ], 1:50,000 or 1:100,000 mouse α-HSC70 (StressGen Bioreagents SPA-817), or 1:2,000 mouse α-mito ATP synthase (MitoScience MS507).

Techniques: Expressing, Control, Western Blot, Membrane, Synthesized, Mutagenesis, Fluorescence, Construct, Confocal Microscopy

Journal: PLoS ONE

Article Title: The Early-Acting Peroxin PEX19 Is Redundantly Encoded, Farnesylated, and Essential for Viability in Arabidopsis thaliana

doi: 10.1371/journal.pone.0148335

Figure Lengend Snippet: (A-E) YFP-PEX19 is mostly cytosolic. Cotyledon epidermal cells from 5-day-old light-grown seedlings carrying various YFP-tagged constructs were imaged using confocal microscopy. YFP directed to the peroxisome (YFP-ECH2) displays punctate fluorescence (A) and ER-directed YFP (ER-YFP-HDEL) displays reticulated fluorescence (B). YFP-PEX19A (D) and YFP-PEX19B (E) fluorescence patterns are neither punctate nor reticulated but resemble untagged YFP fluorescence (C), suggesting cytosolic localization. Each pair of images captures the same cells imaged through the middle (left column) or subcortical region (right column) of the cells. See for corresponding bright-field images. (F) Seedlings relying on YFP-PEX19A or YFP-PEX19B as the sole source of PEX19 respond to IBA similarly to wild-type seedlings. Error bars show standard deviations of mean 8-day-old light-grown root lengths ( n ≥ 9). (G) Expression of YFP-PEX19B confers slight PTS2-processing defects. Protein extracted from 8-day-old light-grown seedlings was separated in triplicate using 10% PAGE and processed for immunoblotting with antibodies recognizing PEX19 or GFP (to detect YFP-PEX19; top and middle panels) and PMDH (bottom panels). Membranes were subsequently probed with α-HSC70 (loading control). The positions of the molecular mass markers (in kDa) are indicted at the left. PMDH is synthesized as a precursor (p) with a cleavable PTS2 signal that is processed into the mature (m) protein in the peroxisome.

Article Snippet: After transfer, membranes were rocked for 1 hour at 4°C in blocking buffer (8% non-fat dry milk [w/v], 20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20) and incubated overnight at 4°C with primary antibodies diluted in blocking buffer: 1:1,000 rabbit α-APX3 [ , ], 1:100 rabbit α-GFP (Clontech 632376), 1:100 rabbit α-PEX5 [ ], 1:800 rabbit α-PEX7 [ ], 1:500 rabbit α-PEX10 [ ], 1:10,000 rabbit α-PEX14 (Agrisera AS08 372), 1:500 or 1:2000 rabbit α-PEX19 (generated and affinity purified by Proteintech Group using a recombinant protein that included the full amino acid sequence of PEX19B), 1:2,000 rabbit α-PMDH2 [ ], 1:5,000 rabbit α-thiolase [ ], 1:50,000 or 1:100,000 mouse α-HSC70 (StressGen Bioreagents SPA-817), or 1:2,000 mouse α-mito ATP synthase (MitoScience MS507).

Techniques: Construct, Confocal Microscopy, Fluorescence, Expressing, Western Blot, Control, Synthesized