Journal: Scientific Reports

Article Title: Intravitreal injection of fibrillin 2 (Fbn2) recombinant protein for therapy of retinopathy in a retina-specific Fbn2 knock-down mouse model

doi: 10.1038/s41598-023-33886-6

Figure Lengend Snippet: Retinal electroretinogram measurements (mean ± standard deviation).

Article Snippet: Primary antibodies against FBN2 (mouse# sc-393968,1:1000, Santa Cruz Biotechnology, USA), TGF (1:2000, Rabbit, ab215715, Abcam Co., Cambridge, MA, USA), LTBP-1 (Rabbit, 1:1000, ab78294, Abcam Co., Cambridge, MA, USA), and β-actin (dilution 1:10,000, Bioss antibodies, Beijing, China) were incubated overnight with the membranes at 4 °C.

Techniques:

Journal: Scientific Reports

Article Title: Intravitreal injection of fibrillin 2 (Fbn2) recombinant protein for therapy of retinopathy in a retina-specific Fbn2 knock-down mouse model

doi: 10.1038/s41598-023-33886-6

Figure Lengend Snippet: Electroretinographical analysis after intravitreal injection with empty carrier, AAV-sh-fbn2 or Fbn2 recombinant protein. NC group: Animals without intervention. AAV-NC group: Animals with an intravitreal injection of AAV empty vector and without any further treatment. AAV-sh-fbn2 group: Animals with an intravitreal injection of AAV-sh-fbn2 and without any further treatment. Fbn2-0.30 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.30 μg. Fbn2-0.75 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.75 μg. Fbn2-1.5 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 1.50 μg. Fbn2-3.0 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 3.00 μg.

Article Snippet: Primary antibodies against FBN2 (mouse# sc-393968,1:1000, Santa Cruz Biotechnology, USA), TGF (1:2000, Rabbit, ab215715, Abcam Co., Cambridge, MA, USA), LTBP-1 (Rabbit, 1:1000, ab78294, Abcam Co., Cambridge, MA, USA), and β-actin (dilution 1:10,000, Bioss antibodies, Beijing, China) were incubated overnight with the membranes at 4 °C.

Techniques: Injection, Recombinant, Plasmid Preparation

Journal: Scientific Reports

Article Title: Intravitreal injection of fibrillin 2 (Fbn2) recombinant protein for therapy of retinopathy in a retina-specific Fbn2 knock-down mouse model

doi: 10.1038/s41598-023-33886-6

Figure Lengend Snippet: Fundus retinal imaging captured by SLO and OCT in NC, AAV-NC, AAV-sh-fbn2, Fbn2-0.30 μg, Fbn2-0.75 μg, Fbn2-1.50 μg and Fbn2-3.0 μg group mice. SLO showed irregular yellow-white exudates in the retina (Black arrow). OCT shows that light reflection in each layer of the retina becomes more irregular and reflectance decreases (Yellow arrow). NC group: Animals without intervention. AAV-NC group: Animals with an intravitreal injection of AAV empty vector and without any further treatment. AAV-sh-fbn2 group: Animals with an intravitreal injection of AAV-sh-fbn2 and without any further treatment. Fbn2-0.30 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.30 μg. Fbn2-0.75 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.75 μg. Fbn2-1.5 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 1.50 μg. Fbn2-3.0 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 3.00 μg.

Article Snippet: Primary antibodies against FBN2 (mouse# sc-393968,1:1000, Santa Cruz Biotechnology, USA), TGF (1:2000, Rabbit, ab215715, Abcam Co., Cambridge, MA, USA), LTBP-1 (Rabbit, 1:1000, ab78294, Abcam Co., Cambridge, MA, USA), and β-actin (dilution 1:10,000, Bioss antibodies, Beijing, China) were incubated overnight with the membranes at 4 °C.

Techniques: Imaging, Injection, Plasmid Preparation, Recombinant

Journal: Scientific Reports

Article Title: Intravitreal injection of fibrillin 2 (Fbn2) recombinant protein for therapy of retinopathy in a retina-specific Fbn2 knock-down mouse model

doi: 10.1038/s41598-023-33886-6

Figure Lengend Snippet: Retina thickness measurements (mean ± standard deviation).

Article Snippet: Primary antibodies against FBN2 (mouse# sc-393968,1:1000, Santa Cruz Biotechnology, USA), TGF (1:2000, Rabbit, ab215715, Abcam Co., Cambridge, MA, USA), LTBP-1 (Rabbit, 1:1000, ab78294, Abcam Co., Cambridge, MA, USA), and β-actin (dilution 1:10,000, Bioss antibodies, Beijing, China) were incubated overnight with the membranes at 4 °C.

Techniques:

Journal: Scientific Reports

Article Title: Intravitreal injection of fibrillin 2 (Fbn2) recombinant protein for therapy of retinopathy in a retina-specific Fbn2 knock-down mouse model

doi: 10.1038/s41598-023-33886-6

Figure Lengend Snippet: Retinal thickness imaging captured by OCT in NC, AAV-NC, AAV-sh-fbn2, Fbn2-0.30 μg, Fbn2-0.75 μg, Fbn2-1.50 μg and Fbn2-3.0 μg group mice. ( A ). Retinal thickness was measured at 3000 μm from optic disc (inferior, nasal, temporal, and superior) ( B ).NC group: Animals without intervention. AAV-NC group: Animals with an intravitreal injection of AAV empty vector and without any further treatment. AAV-sh-fbn2 group: Animals with an intravitreal injection of AAV-sh-fbn2 and without any further treatment. Fbn2-0.30 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.30 μg. Fbn2-0.75 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.75 μg. Fbn2-1.5 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 1.50 μg. Fbn2-3.0 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 3.00 μg.

Article Snippet: Primary antibodies against FBN2 (mouse# sc-393968,1:1000, Santa Cruz Biotechnology, USA), TGF (1:2000, Rabbit, ab215715, Abcam Co., Cambridge, MA, USA), LTBP-1 (Rabbit, 1:1000, ab78294, Abcam Co., Cambridge, MA, USA), and β-actin (dilution 1:10,000, Bioss antibodies, Beijing, China) were incubated overnight with the membranes at 4 °C.

Techniques: Imaging, Injection, Plasmid Preparation, Recombinant

Journal: Scientific Reports

Article Title: Intravitreal injection of fibrillin 2 (Fbn2) recombinant protein for therapy of retinopathy in a retina-specific Fbn2 knock-down mouse model

doi: 10.1038/s41598-023-33886-6

Figure Lengend Snippet: Axial length measured by OCT. NC group: Animals without intervention. AAV-NC group: Animals with an intravitreal injection of AAV empty vector and without any further treatment. AAV-sh-fbn2 group: Animals with an intravitreal injection of AAV-sh-fbn2 and without any further treatment. Fbn2-0.30 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.30 μg. Fbn2-0.75 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.75 μg. Fbn2-1.5 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 1.50 μg. Fbn2-3.0 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 3.00 μg. NC group: Animals without intervention. AAV-NC group: Animals with an intravitreal injection of AAV empty vector and without any further treatment. AAV-sh-fbn2 group: Animals with an intravitreal injection of AAV-sh-fbn2 and without any further treatment. Fbn2-0.30 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.30 μg. Fbn2-0.75 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.75 μg. Fbn2-1.5 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 1.50 μg. Fbn2-3.0 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 3.00 μg.

Article Snippet: Primary antibodies against FBN2 (mouse# sc-393968,1:1000, Santa Cruz Biotechnology, USA), TGF (1:2000, Rabbit, ab215715, Abcam Co., Cambridge, MA, USA), LTBP-1 (Rabbit, 1:1000, ab78294, Abcam Co., Cambridge, MA, USA), and β-actin (dilution 1:10,000, Bioss antibodies, Beijing, China) were incubated overnight with the membranes at 4 °C.

Techniques: Injection, Plasmid Preparation, Recombinant

Journal: Scientific Reports

Article Title: Intravitreal injection of fibrillin 2 (Fbn2) recombinant protein for therapy of retinopathy in a retina-specific Fbn2 knock-down mouse model

doi: 10.1038/s41598-023-33886-6

Figure Lengend Snippet: Measurement of fbn2, Tgf-β1 and LTBP-1 expression at mRNA levels in the retina of mice in NC, AAV-NC, AAV-sh-fbn2, Fbn2-0.30 μg, Fbn2-0.75 μg, Fbn2-1.50 μg and Fbn2-3.0 μg groups (all groups n = 9). Data are shown as mean ± SEM. * P < 0.05 all other groups versus AAV-NC group; ** P < 0.001 all other groups versus AAV-NC group; # P < 0.05 all other groups versus AAV-sh-fbn2 group NC group: Animals without intervention. AAV-NC group: Animals with an intravitreal injection of AAV empty vector and without any further treatment. AAV-sh-fbn2 group: Animals with an intravitreal injection of AAV-sh-fbn2 and without any further treatment. Fbn2-0.30 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.30 μg. Fbn2-0.75 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.75 μg. Fbn2-1.5 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 1.50 μg. Fbn2-3.0 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 3.00 μg.

Article Snippet: Primary antibodies against FBN2 (mouse# sc-393968,1:1000, Santa Cruz Biotechnology, USA), TGF (1:2000, Rabbit, ab215715, Abcam Co., Cambridge, MA, USA), LTBP-1 (Rabbit, 1:1000, ab78294, Abcam Co., Cambridge, MA, USA), and β-actin (dilution 1:10,000, Bioss antibodies, Beijing, China) were incubated overnight with the membranes at 4 °C.

Techniques: Expressing, Injection, Plasmid Preparation, Recombinant

Journal: Scientific Reports

Article Title: Intravitreal injection of fibrillin 2 (Fbn2) recombinant protein for therapy of retinopathy in a retina-specific Fbn2 knock-down mouse model

doi: 10.1038/s41598-023-33886-6

Figure Lengend Snippet: Elisa measurement of Fbn2, Tgf-β1 and LTBP-1 expression at protein levels in retina of mice in NC, AAV-NC, AAV-sh-fbn2, Fbn2-0.30 μg, Fbn2-0.75 μg, Fbn2-1.50 μg and Fbn2-3.0 μg group (all groups n = 9). Data are shown as mean ± SEM. * P < 0.05 all other groups versus AAV-NC group; ** P < 0.001 all other groups versus AAV-NC group; # P < 0.05 all other groups versus AAV-sh-fbn2 group. NC group: Animals without intervention. AAV-NC group: Animals with an intravitreal injection of AAV empty vector and without any further treatment. AAV-sh-fbn2 group: Animals with an intravitreal injection of AAV-sh-fbn2 and without any further treatment. Fbn2-0.30 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.30 μg. Fbn2-0.75 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.75 μg. Fbn2-1.5 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 1.50 μg. Fbn2-3.0 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 3.00 μg.

Article Snippet: Primary antibodies against FBN2 (mouse# sc-393968,1:1000, Santa Cruz Biotechnology, USA), TGF (1:2000, Rabbit, ab215715, Abcam Co., Cambridge, MA, USA), LTBP-1 (Rabbit, 1:1000, ab78294, Abcam Co., Cambridge, MA, USA), and β-actin (dilution 1:10,000, Bioss antibodies, Beijing, China) were incubated overnight with the membranes at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Injection, Plasmid Preparation, Recombinant

Journal: Scientific Reports

Article Title: Intravitreal injection of fibrillin 2 (Fbn2) recombinant protein for therapy of retinopathy in a retina-specific Fbn2 knock-down mouse model

doi: 10.1038/s41598-023-33886-6

Figure Lengend Snippet: Measurement of fbn2, Tgf-β1 and LTBP-1 expression at protein levels using Western blot in the retina of mice in NC, AAV-NC, AAV-sh-fbn2 and Fbn2-0.75 μg group (all groups n = 9). Data are shown as mean ± SEM. ** P < 0.001 all other groups versus AAV-NC group; # P < 0.05 all other groups versus AAV-sh-fbn2 group. Original blots are presented in Supplementary Fig. – . A: NC group, B: AAV-NC group, C: AAV-sh-fbn2 group, D: Fbn2-0.75 μg group. (** P < 0.001 compared with the AAV-NC group; # P < 0.05 compared with the AAV-sh-fbn2 group). NC group: Animals without intervention. AAV-NC group: Animals with an intravitreal injection of AAV empty vector and without any further treatment. AAV-sh-fbn2 group: Animals with an intravitreal injection of AAV-sh-fbn2 and without any further treatment. Fbn2-0.30 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.30 μg. Fbn2-0.75 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 0.75 μg. Fbn2-1.5 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 1.50 μg. Fbn2-3.0 μg group: Animals with an intravitreal injection of AAV-sh-fbn2, followed by an intravitreal injection of fbn2 recombinant protein in a dose of 3.00 μg.

Article Snippet: Primary antibodies against FBN2 (mouse# sc-393968,1:1000, Santa Cruz Biotechnology, USA), TGF (1:2000, Rabbit, ab215715, Abcam Co., Cambridge, MA, USA), LTBP-1 (Rabbit, 1:1000, ab78294, Abcam Co., Cambridge, MA, USA), and β-actin (dilution 1:10,000, Bioss antibodies, Beijing, China) were incubated overnight with the membranes at 4 °C.

Techniques: Expressing, Western Blot, Injection, Plasmid Preparation, Recombinant

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: Plant physiology

Article Title: Arabidopsis FIBRILLIN6 influences carotenoid biosynthesis by directly promoting phytoene synthase activity.

doi: 10.1093/plphys/kiad613

Figure Lengend Snippet: Figure 2. FBN6 and PSY co-localize in chloroplasts. N. benthamiana leaves were co-agroinfiltrated with constructs encoding FBN6-RFP and PSY-GFP, and 4 d later, samples were taken for confocal and immunoblot analyses. A) Representative confocal microscopy images of a leaf section transiently expressing the indicated fluorescent fusion proteins. All panels in the same column show the same field. RFP fluorescence is shown in the top panels, GFP fluorescence in the second row, and chlorophyll autofluorescence in the third row. Bottom panels show a merge of the 3 fluorescence channels. Right panels are a magnification of the area boxed in white in the left panels. Bars, 5 µm. B) Immunoblot analysis of the accumulation of FBNs iden- tified using antibodies against Arabidopsis PG-associated FBN1a and FBN2 proteins in leaves expressing the indicated constructs or a GFP control (−/−). Quantification was made by relativizing each band intensity to the one obtained in the control. Representative results are shown in the lower panels. Barplot shows the mean ± SD values of quantitative data from n = 3 leaves.

Article Snippet: Blots were incubated with available antibodies against FBN1a and FBN2 (Morelli et al. 2023) diluted 1:5,000 or commercial antibodies against GFP (Invitrogen, 1:2,000), Myc (Calbiochem, 1:5,000), HA (Sigma, 1:1,000) and 6× His (Proteintech, 1:5,000) epitopes.

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