Journal: Journal of Experimental Botany

Article Title: Arabidopsis fibrillin 1-2 subfamily members exert their functions via specific protein–protein interactions

doi: 10.1093/jxb/erab452

Figure Lengend Snippet: Isolation of the fbn2 knockout mutant. (A) Schematic illustration of the genomic DNA fragment containing the FBN2 gene. The T-DNA insertion in the first exon of FBN2 found in the SALK_124590 line is shown. The primers used in the analysis of wild-type (WT) or mutant FBN2 alleles are indicated. (B) PCR analysis of a homozygous fbn2 mutant plant. Only the mutant allele is detected. (C) Immunoblot analysis of leaf extracts of WT and fbn2 mutant plants using anti-FBN2 antibodies.

Article Snippet: The pellet was discarded and the supernatant was subjected to co-immunoprecipitation (Co-IP) analysis using a polyclonal specific antibody against the whole FBN2 protein (produced by GENSCRIPT) and a Dynabeads Co-Immunoprecipitation Kit (Life Technologies, http://lifetechnologies.com ) following the manufacturer’s instructions.

Techniques: Isolation, Knock-Out, Mutagenesis, Western Blot

Journal: Journal of Experimental Botany

Article Title: Arabidopsis fibrillin 1-2 subfamily members exert their functions via specific protein–protein interactions

doi: 10.1093/jxb/erab452

Figure Lengend Snippet: Localization of FBN2. (A) Full-length FBN2 or FBN4 cDNAs were fused to GFP and transiently expressed in N. benthamiana leaves. Fluorescence was monitored by confocal microscopy. The GFP fluorescence, chlorophyll autofluorescence, and merged images are shown. (B) Chloroplasts isolated from Arabidopsis rosette leaves were disrupted and the soluble (S) and membrane fractions (M) were isolated by ultracentrifugation at 100000 g for 1h at 4 °C. The pellet (membrane fraction) was resuspended in the same volume as the supernatant and 20 µg of protein from each fraction was loaded on to SDS-PAGE gels. The proteins were blotted on to a PVDF filter and hybridized with specific antibodies against FBN2, FBN4, plastidial glutamine synthetase (GS) (a marker of stromal protein), and psbA (a marker of thylakoid membranes).

Article Snippet: The pellet was discarded and the supernatant was subjected to co-immunoprecipitation (Co-IP) analysis using a polyclonal specific antibody against the whole FBN2 protein (produced by GENSCRIPT) and a Dynabeads Co-Immunoprecipitation Kit (Life Technologies, http://lifetechnologies.com ) following the manufacturer’s instructions.

Techniques: Fluorescence, Confocal Microscopy, Isolation, Membrane, SDS Page, Marker

Journal: Journal of Experimental Botany

Article Title: Arabidopsis fibrillin 1-2 subfamily members exert their functions via specific protein–protein interactions

doi: 10.1093/jxb/erab452

Figure Lengend Snippet: Anthocyanin accumulation in fbn mutants. (A) Knockout mutants fbn2 , fbn1a-1b , and fbn1a-1b-2 and WT plants were sown in soil and cultivated under normal conditions for 3 weeks. After this period, the plants were subjected to stress conditions (600 µmol m −2 s −1 light intensity and 10 °C temperature). Twelve rosette leaves from four plants per line were harvested after 0, 1, and 3 weeks of treatment, and the concentrations of anthocyanins were determined. (B) Plants cultivated under normal conditions were subjected to stress as described in (A) for 1 week with or without the addition of 2mM JA every 3 d, and the concentrations of anthocyanins were determined. In both panels, the values are presented as means ±SD. Two-way ANOVA was performed using PRISM software version 6.0. Tukey’s HSD test was used as a post hoc test. Significant differences ( P <0.01) between mean values are indicated with different letters.

Article Snippet: The pellet was discarded and the supernatant was subjected to co-immunoprecipitation (Co-IP) analysis using a polyclonal specific antibody against the whole FBN2 protein (produced by GENSCRIPT) and a Dynabeads Co-Immunoprecipitation Kit (Life Technologies, http://lifetechnologies.com ) following the manufacturer’s instructions.

Techniques: Knock-Out, Software

Journal: Journal of Experimental Botany

Article Title: Arabidopsis fibrillin 1-2 subfamily members exert their functions via specific protein–protein interactions

doi: 10.1093/jxb/erab452

Figure Lengend Snippet: PSII performance of fbn plants. The plants were sown in soil, cultivated under normal conditions for 3 weeks, and then stressed for 1 week with a combination of moderate high light and low temperature as described in . (A) Representative false-colour images of WT, fbn2 , fbn1a-1b , and fbn1a-1b-2 plants at 0 d, 1 d, and 1 week of stress. The images represent the maximum PSII quantum yield ( F v / F m ). The last column shows the phenotypes of plants after 1 week of stress. (B) Six fully expanded leaves from two plants per line at 0h, 24h, and 1 week of stress were used to determine the F v / F m values. (C) Concentrations of MDA in the leaves of 3-week-old plants subjected to high-light and cold stresses for 0h and 1 week. Four plants (three fully expanded leaves per plant) per line were used to determine the concentrations of MDA at each time point. In (B) and (C) the values are presented as means ±SD. PRISM software (version 6.0) was used to carry out a two-way ANOVA with Tukey’s post hoc test. Significant differences ( P <0.01) between mean values are indicated with different letters.

Article Snippet: The pellet was discarded and the supernatant was subjected to co-immunoprecipitation (Co-IP) analysis using a polyclonal specific antibody against the whole FBN2 protein (produced by GENSCRIPT) and a Dynabeads Co-Immunoprecipitation Kit (Life Technologies, http://lifetechnologies.com ) following the manufacturer’s instructions.

Techniques: Software

Journal: Journal of Experimental Botany

Article Title: Arabidopsis fibrillin 1-2 subfamily members exert their functions via specific protein–protein interactions

doi: 10.1093/jxb/erab452

Figure Lengend Snippet: Accumulation of anthocyanins and F v / F m of fbn2 plants transformed with the FBN2 gene. (A) WT, fbn2 , and two transgenic plants transformed with a genomic DNA fragment containing the FBN2 gene and 1kb of its promoter region (plants T4.5 and T5.6) were cultivated in a growth chamber under normal conditions and then subjected to stress conditions as described in . Twelve rosette leaves from four plants per line were harvested after 0, 1, and 3 weeks of treatment, and the concentrations of anthocyanins were determined. (B) WT, T4.5, and T5.6 plants grown under normal conditions were subjected to high-light and cold stresses, and the F v / F m values of six fully expanded leaves from two plants per line were determined at 0 d, 1 d, and 1 week of treatment using an IMAGING-PAM device. In both panels, the values are presented as means ±SD. PRISM software (version 6.0) was used to carry out a two-way ANOVA with Tukey’s post hoc test analysis. Significant differences ( P <0.01) between mean values are indicated with different letters.

Article Snippet: The pellet was discarded and the supernatant was subjected to co-immunoprecipitation (Co-IP) analysis using a polyclonal specific antibody against the whole FBN2 protein (produced by GENSCRIPT) and a Dynabeads Co-Immunoprecipitation Kit (Life Technologies, http://lifetechnologies.com ) following the manufacturer’s instructions.

Techniques: Transformation Assay, Transgenic Assay, Imaging, Software

Journal: Journal of Experimental Botany

Article Title: Arabidopsis fibrillin 1-2 subfamily members exert their functions via specific protein–protein interactions

doi: 10.1093/jxb/erab452

Figure Lengend Snippet: In vivo interaction of FBN2–FBN1a, FBN2–FBN1b, FBN2–FBN2, FBN2–FBN4, and FBN1a–FBN4. cDNAs encoding the full-length FBN1a or FBN2 proteins were fused to the N-terminal half of YFP and co-transformed into N. benthamiana leaves together with cDNAs encoding FBN2, FBN1a, FBN1b, or FBN4 fused to the C-terminal moiety of CFP. The images show the YFP/CFP (BiFC) fluorescence, the chlorophyll autofluorescence, and the merged images.

Article Snippet: The pellet was discarded and the supernatant was subjected to co-immunoprecipitation (Co-IP) analysis using a polyclonal specific antibody against the whole FBN2 protein (produced by GENSCRIPT) and a Dynabeads Co-Immunoprecipitation Kit (Life Technologies, http://lifetechnologies.com ) following the manufacturer’s instructions.

Techniques: In Vivo, Transformation Assay, Fluorescence

Journal: Journal of Experimental Botany

Article Title: Arabidopsis fibrillin 1-2 subfamily members exert their functions via specific protein–protein interactions

doi: 10.1093/jxb/erab452

Figure Lengend Snippet: In vivo interaction of FBN2–AOS, FBN1a–AOS, and FBN1b–AOS. cDNAs encoding the full-length FBN1a, FBN1b, or FBN2 proteins were fused to the N-terminal half of YFP and co-transformed into N. benthamiana leaves together with cDNA encoding AOS fused to the C-terminal moiety of CFP. Images show the YFP/CFP (BiFC) fluorescence, the chlorophyll autofluorescence, and the merged images.

Article Snippet: The pellet was discarded and the supernatant was subjected to co-immunoprecipitation (Co-IP) analysis using a polyclonal specific antibody against the whole FBN2 protein (produced by GENSCRIPT) and a Dynabeads Co-Immunoprecipitation Kit (Life Technologies, http://lifetechnologies.com ) following the manufacturer’s instructions.

Techniques: In Vivo, Transformation Assay, Fluorescence

Journal: Journal of Experimental Botany

Article Title: Arabidopsis fibrillin 1-2 subfamily members exert their functions via specific protein–protein interactions

doi: 10.1093/jxb/erab452

Figure Lengend Snippet: Schematic model of the arrangement of FBNs1-2 subgroup proteins on the surface of PGs. FBN2 (red) may form homodimers or heterodimers with FBN1a (light brown) or FBN1b (dark brown). We have previously shown that FBN1a and FBN1b may form hetero-oligomers ( Gámez-Arjona et al , 2014 a ). These interactions allow the formation of a FBNs1-2-based network around the surface of PGs. Other proteins, such as those described in , associate with PGs via interactions with these FBNs. The degree of functional redundancy between these FBNs has not been characterized and might vary for each PG-associated protein. Their elimination would affect the localization and function of some PG-associated proteins. The functions of other FBNs associated with PGs (FBN4, FBN7a, FBN7b, and FBN8, indicated in light blue) have not been determined yet.

Article Snippet: The pellet was discarded and the supernatant was subjected to co-immunoprecipitation (Co-IP) analysis using a polyclonal specific antibody against the whole FBN2 protein (produced by GENSCRIPT) and a Dynabeads Co-Immunoprecipitation Kit (Life Technologies, http://lifetechnologies.com ) following the manufacturer’s instructions.

Techniques: Functional Assay

Journal: Pulmonary Circulation

Article Title: Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs

doi: 10.1086/681272

Figure Lengend Snippet: Prx1 regulates transforming growth factor (TGF)–β signaling in embryonic lungs. A, Immunohistochemical staining for TGF-β1 and TGF-β2 in lung sections derived from E15.5 (top) and D0 (bottom) Prx1WT (left) and Prx1null (right) mice. Brown indicates positive staining of antibodies. While TGF-β localization is restricted to the developing vessel area (arrows) in Prx1WT lungs, in Prx1null lungs TGF-β expression is observed in areas other than the vessel wall vicinity (arrowheads). Hematoxylin staining (blue) is used for nuclear counterstaining. Scale bar = 40 μm. B, Western blotting for TGF-β1 protein expression in E17.5 lungs from Prx1WT and Prx1null mice. C, Real-time polymerase chain reaction (PCR) for TGF-β1 messenger RNA (mRNA) expression in E17.5 lungs from Prx1WT and Prx1null mice. D, Western blotting for phosphorylated Smad2 (P-Smad2; top) and total Smad 2/3 (middle) in E17.5 lungs from Prx1WT and Prx1null mice. E, Real-time PCR for plasminogen activator inhibitor type 1 (PAI-1) mRNA expression in E17.5 lungs from Prx1WT and Prx1null mice. For Western immunoblotting (B, D), β-actin was used as a loading control. For mRNA expression by real-time PCR (C, E), target gene expression was normalized to GAPDH. Two asterisks indicate P < 0.01. WT: wild type.

Article Snippet: After being incubated with blocking buffer, membranes were incubated with primary antibodies using the following: Fbn1 and Fbn2 (gift of R. Mecham), tropoelastin (Abcam), TGF-β1 (Promega), phosphorylated Smad2 (Cell Signaling Technology, Danvers, MA), and total Smad2/3 (Cell Signaling Technology).

Techniques: Immunohistochemical staining, Staining, Derivative Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Pulmonary Circulation

Article Title: Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs

doi: 10.1086/681272

Figure Lengend Snippet: 10T1/2 cell differentiation to a smooth muscle phenotype is transforming growth factor (TGF)–β dependent. A, Immunostaining for α–smooth muscle actin (SMA), γ-SMA, SM22α, and smooth muscle myosin heavy chain (SMMHC; all positive reactions are green) in 10T1/2 cells, with or without TGF-β1 treatment, in the presence or absence of a TGF-β type I receptor (TGF-βRI) inhibitor (LY364947). Scale bar = 10 μm. B, α- and γ-SMA messenger RNA expression in 10T1/2 cells after stimulation with TGF-β1, with or without LY364947. Plasminogen activator inhibitor type 1 (PAI-1) was used as a positive control for canonical TGF-β signaling activation. Signals were normalized to GAPDH. C, Western immunoblotting for α- and γ-SMA expression in 10T1/2 cells after stimulation with TGF-β1, with or without a TGF-βRI inhibitor (SB431542). Phosphorylation of Smad2 (P-Smad2) was examined as a marker of canonical TGF-β signaling. GAPDH was used as a loading control.

Article Snippet: After being incubated with blocking buffer, membranes were incubated with primary antibodies using the following: Fbn1 and Fbn2 (gift of R. Mecham), tropoelastin (Abcam), TGF-β1 (Promega), phosphorylated Smad2 (Cell Signaling Technology, Danvers, MA), and total Smad2/3 (Cell Signaling Technology).

Techniques: Cell Differentiation, Immunostaining, RNA Expression, Positive Control, Activation Assay, Western Blot, Expressing, Marker

Journal: Human Molecular Genetics

Article Title: ADAMTS10-mediated tissue disruption in Weill–Marchesani syndrome

doi: 10.1093/hmg/ddy276

Figure Lengend Snippet: Perturbed skeletal muscle homeostasis in WMS ADAMTS10 S236X/S236X muscle. (A) TEM of the 3-month-old WT and HOM muscle tissue showing enlarged mitochondria in the latter, scale bar = 1 μm. (B) Quantification of size and number of mitochondria (one mouse 4 areas). (C) Western blots of phospho-p38 (Pp38) and p38 protein levels in the 4-week-old skeletal muscle lysates showing increased MAPK signalling in the WMS muscle. Normalized quantifications, n = 3. Statistical significance was calculated using two-tail unpaired Student’s test in GraphPad Prism V6. Asterisk indicate P -values where **** P -value ≤ 0.0001.

Article Snippet: Primary antibodies used for immunofluorescence microscopy and western blotting were ADAMTS10 (custom synthesized pro-domain specific rabbit, Biomatik, 1:50), rat anti-ZO-1 (Millipore; T11, 1:200), rabbit anti-Laminin (Abcam, ab11575, 1:100), rabbit anti-PECAM (Abcam, ab 28364, 1:100), rat anti-BrdU (Sigma-Aldrich, RPN201,1:200), goat anti-MAGP1 (Santa Cruiz, sc-166075, 1:200), rabbit anti-Pp38 antibody (Cell Signalling 9211, 1:500) and rabbit anti-p38 antibody (Cell Signalling 8690, 1:500), rabbit anti-pSMAD1/5/8 (Millipore, AB3848-I, 1:500), rabbit anti-SMAD1 (Cell Signalling 9743, 1:500), rabbit anti-Fbn1 and rabbit anti-Fbn2 (1:500).

Techniques: Western Blot

Journal: Human Molecular Genetics

Article Title: ADAMTS10-mediated tissue disruption in Weill–Marchesani syndrome

doi: 10.1093/hmg/ddy276

Figure Lengend Snippet: Perturbed skeletal muscle homeostasis in WMS ADAMTS10 S236X/S236X muscle. (A) TEM of the 3-month-old WT and HOM muscle tissue showing enlarged mitochondria in the latter, scale bar = 1 μm. (B) Quantification of size and number of mitochondria (one mouse 4 areas). (C) Western blots of phospho-p38 (Pp38) and p38 protein levels in the 4-week-old skeletal muscle lysates showing increased MAPK signalling in the WMS muscle. Normalized quantifications, n = 3. Statistical significance was calculated using two-tail unpaired Student’s test in GraphPad Prism V6. Asterisk indicate P -values where **** P -value ≤ 0.0001.

Article Snippet: Primary antibodies used for immunofluorescence microscopy and western blotting were ADAMTS10 (custom synthesized pro-domain specific rabbit, Biomatik, 1:50), rat anti-ZO-1 (Millipore; T11, 1:200), rabbit anti-Laminin (Abcam, ab11575, 1:100), rabbit anti-PECAM (Abcam, ab 28364, 1:100), rat anti-BrdU (Sigma-Aldrich, RPN201,1:200), goat anti-MAGP1 (Santa Cruiz, sc-166075, 1:200), rabbit anti-Pp38 antibody (Cell Signalling 9211, 1:500) and rabbit anti-p38 antibody (Cell Signalling 8690, 1:500), rabbit anti-pSMAD1/5/8 (Millipore, AB3848-I, 1:500), rabbit anti-SMAD1 (Cell Signalling 9743, 1:500), rabbit anti-Fbn1 and rabbit anti-Fbn2 (1:500).

Techniques: Western Blot