Journal: Communications Biology

Article Title: Arabidopsis MYB47 and MYB95 transcription factors regulate jasmonate-inducible ER-body formation

doi: 10.1038/s42003-025-08863-6

Figure Lengend Snippet: a Confocal laser scanning microscope images of cotyledons from 10-day-old stable transgenic plants in which ER and ER bodies are visualized with GFP. The GFP signal shows the ER and ER bodies. b Accumulation of BGLU23 and NAI2 proteins in 10-day-old Arabidopsis seedlings. The extracted proteins from seedlings were subjected to immunoblot analysis with anti-BGLU23 antibody or anti-NAI2 antibody. The bands correspond to full length (upper) and partially degraded (lower) NAI2 proteins are shown with arrowheads. Coomassie brilliant blue (CBB) staining shows the RuBisCo large subunit (RbcL) used as a loading control.

Article Snippet: A protein complex containing the GFP-MYB fusion proteins was co-immunoprecipitated using the μMACS GFP isolation kit (Miltenyi Biotec, Germany).

Techniques: Laser-Scanning Microscopy, Transgenic Assay, Western Blot, Staining, Control

Journal: Communications Biology

Article Title: Arabidopsis MYB47 and MYB95 transcription factors regulate jasmonate-inducible ER-body formation

doi: 10.1038/s42003-025-08863-6

Figure Lengend Snippet: a Interaction between MYB and MYC proteins in the yeast two-hybrid assay. Constructs expressing the DNA-binding domain (BD)-MYB and activation domain (AD)-MYC fusion proteins were co-expressed in yeast, and protein–protein interactions were examined by measuring β-galactosidase activity. Empty vectors were used as a negative control. Error bars indicate SE ( n = 3 independent experiments). Different lowercase letters indicate significant differences ( p < 0.05; Tukey’s test). AU, arbitrary unit. b Interaction between MYB and MYC proteins in planta . Constructs expressing the GFP-MYB and RFP-MYC fusion proteins were transiently expressed in N. benthamiana leaves, and input and immunoprecipitated protein complexes were detected by immunoblot analysis. Asterisks indicate nonspecific cross-reactive bands detected using anti-GFP and anti-RFP antibodies.

Article Snippet: A protein complex containing the GFP-MYB fusion proteins was co-immunoprecipitated using the μMACS GFP isolation kit (Miltenyi Biotec, Germany).

Techniques: Y2H Assay, Construct, Expressing, Binding Assay, Activation Assay, Protein-Protein interactions, Activity Assay, Negative Control, Immunoprecipitation, Western Blot

Journal: Cellular Microbiology

Article Title: PERP, a host tetraspanning membrane protein, is required for S almonella ‐induced inflammation

doi: 10.1111/cmi.12406

Figure Lengend Snippet: SipA interacting partner candidates

Article Snippet: The dual‐hunter split ubiquitin Y2H kit was used (Dualsystems Biotech AG).

Techniques: Activation Assay

Journal: Frontiers in Plant Science

Article Title: Arabidopsis PCNAs form complexes with selected D-type cyclins

doi: 10.3389/fpls.2015.00516

Figure Lengend Snippet: Analysis of interactions between Arabidopsis PCNA1/2 and D-type cyclins using the split-uiquitin Y2H system . The interactions were tested in the following combinations. (A) PCNA1 (bait)/D-type cyclins (prey), (B) PCNA2 (bait)/D-type cyclins (prey), and (C) D-type cyclins (bait)/PCNA1/2 (prey). The transformed yeast cells were plated on either an SC-Leu-Trp control solid medium or an SC-Leu-Trp-His selection solid medium supplemented with 10 mM 3-aminotriazol (3-AT). For the beta-galactosidase assay, the yeast transformants were grown on a nitrocellulose filter placed on the surface of a YPAD solid medium followed by incubation with X-gal. The results are representative of three independently repeated experiments.

Article Snippet: The pDHB1 bait and pPR3-N prey plasmids used in the split-ubiquitin Y2H system transactivating starter kit (MoBiTec) were reconstructed into Gateway-compatible vectors.

Techniques: Transformation Assay, Control, Selection, β-Gal Assay, Incubation

Journal: Journal of Cachexia, Sarcopenia and Muscle

Article Title: Myopalladin promotes muscle growth through modulation of the serum response factor pathway

doi: 10.1002/jcsm.12486

Figure Lengend Snippet: The MYPN Ig3 domain binds to myocardin‐related transcription factors (MRTFs). (A, B) Yeast‐two hybrid (Y2H) assay showing binding of the MYPN Ig3 domain to MRTF‐A residue 347–611, comprising the SAP and LZ domains. MYPN binds less strongly to MRTF‐B. (A) The domain structure of MRTF‐A is shown with the constructs that were tested for binding to MYPN in the Y2H system below. RPEL, conserved N‐terminal domain; ++, basic domain; Q, glutamine‐rich domain; SAP, SRF‐A/B‐Acinus‐PIAS domain, LZ, leucine zipper‐like domain, TAD, transactivation domain. (B) Culture plate with the different combinations of Y2H cotransformations as shown in the table on the left. (C) Co‐immunoprecipitation (Co‐IP) assay with anti‐HA antibody after transfection of HEK293 cells with HA‐tagged MYPN constructs and FLAG‐tagged MRTF‐A, confirming the binding of MYPN to MRTF‐A. The experiment was repeated three times. (D) Immunofluorescence analysis of TA muscle electroporated with PmCherry‐N1‐MYPN (red) and stained for MRTF‐A (green), showing colocalization in the sarcomere and nucleus. Nuclei are visualized by DAPI (blue).

Article Snippet: pGBKT‐BD bait and pGADT7‐AD prey vectors (Clontech Laboratories) containing cDNA encoding the proteins of interest were cotransformed into the Y2H Gold yeast strain (Clontech Laboratories) using the Frozen‐EZ Yeast Transformation II TM kit (Zymo Research) following the manufacturer's instructions.

Techniques: Y2H Assay, Binding Assay, Construct, Co-Immunoprecipitation Assay, Transfection, Immunofluorescence, Staining

Journal: Biomolecules

Article Title: The Involvement of the Banana F-Box Protein MaEBF1 in Regulating Chilling-Inhibited Starch Degradation through Interaction with a MaNAC67-Like Protein

doi: 10.3390/biom9100552

Figure Lengend Snippet: The expression profiles of genes related to starch degradation in Fenjiao fruit pulp under different storage conditions. The expression patterns of MaEBF1 ( A ) and starch degradation genes ( B – L ) after different storage conditions and ripening process. The expression levels of each gene at different days are relative to that of 0 d (freshly harvested fruit), which was set as 1. Each value represents the means of biological triplicates (mean ± SD). Different letters above the bars indicate significant differences at the 5% level. Fruit were stored at 25 °C, 11 °C, and 7 °C for 6 or 12 days, removed to 25 °C, and treated with ethephon.

Article Snippet: The MaEBF1-GST protein was purified using the GST Purification Kit (Clontech, Cat. No. 635619, Fitchburg, WI, USA) and the MaNAC67-like-His protein was purified using the Ni-NTA His Purification Kit (Clontech, Cat. No. 635658).

Techniques: Expressing

Journal: Biomolecules

Article Title: The Involvement of the Banana F-Box Protein MaEBF1 in Regulating Chilling-Inhibited Starch Degradation through Interaction with a MaNAC67-Like Protein

doi: 10.3390/biom9100552

Figure Lengend Snippet: Interaction between MaEBF1 and MaNAC67-like proteins. ( A , B ) Y2H assay showing the interaction of MaEBF1 with MaNAC67-like. Diagram of the different constructs used in this experiment ( A ); the Y2H yeast strains were co-transformed with MaEBF1 + MaNAC67-like. Yeast cells grew on SD/-Leu-Trp-Ade-His in the presence of 125 μm Aureobasidin A, and blue plaques served as a positive interaction with chromogenic substrate X-α-gal ( B ); ( C ) the expression profile of MaNAC67-like during cold storage and ripening process; ( D ) the subcellular localization of MaEBF1 and MaNAC67-like proteins. ( E ) In vitro pull-down assay of the interaction between MaEBF1 with MaNAC67-like. ( F ) BiFC analysis of the interactions of MaEBF1 with MaNAC67-like in tobacco leaf epidermal cells. Bars, 50 μm.

Article Snippet: The MaEBF1-GST protein was purified using the GST Purification Kit (Clontech, Cat. No. 635619, Fitchburg, WI, USA) and the MaNAC67-like-His protein was purified using the Ni-NTA His Purification Kit (Clontech, Cat. No. 635658).

Techniques: Y2H Assay, Construct, Transformation Assay, Expressing, In Vitro, Pull Down Assay

Journal: Biomolecules

Article Title: The Involvement of the Banana F-Box Protein MaEBF1 in Regulating Chilling-Inhibited Starch Degradation through Interaction with a MaNAC67-Like Protein

doi: 10.3390/biom9100552

Figure Lengend Snippet: Transcriptional activation of starch degradation genes by MaEBF1 and MaNAC67-like in the transient expression system. The promoters of MaBAM6, MaMEX1, and MaSEX4 were ligated into a pGreenII 0800-LUC double-reporter vector ( A ), while MaNAC67-like was ligated into the pGreenII 62-SK vector as an effector ( B ). The transcriptional activity of MaNAC67-like on the starch degradation-related gene promoter of MaBAM3 , MaBAM4 , MaBAM6 , MaBAM7 , MaBAM8 , MaPWD1 , MaGWD1 , MaAMY3 , MaISA2 , MaSEX4 , and MaMEX1 ( C ). The ratio of LUC to REN of the empty vector plus promoter vector was used as a calibrator (set as 1). ( D – F ) Transcriptional activity of MaEBF1, MaNAC67-like, and MaNAC67-like + MaEBF1 on the activities of promoter of MaBAM6 ( D ), MaMEX1 ( E ), and MaSEX4 ( F ). Each value was calculated from the means of six biological replicates. The values represent the means ± SE.

Article Snippet: The MaEBF1-GST protein was purified using the GST Purification Kit (Clontech, Cat. No. 635619, Fitchburg, WI, USA) and the MaNAC67-like-His protein was purified using the Ni-NTA His Purification Kit (Clontech, Cat. No. 635658).

Techniques: Activation Assay, Expressing, Plasmid Preparation, Activity Assay