Journal: bioRxiv

Article Title: A novel repressor-activator-competitor module comprising C 2 H 2 zinc finger and NAC transcription factors regulates rice grain development

doi: 10.1101/2024.07.15.603577

Figure Lengend Snippet: (A) Schematic representation of ZOS1-15 and its truncated forms. ZF1 and ZF2 represent C 2 H 2 zinc fingers, DLN represents the repression motif, ΔN, ΔC and ΔDLN represent deletions of the respective regions. Diagram is not drawn to scale. (B) Y2H assays show homodimerization of ZOS1-15 and (C) its interacting partners, MED14_1, HDT702, OsTPL and ONAC024. _AD and _BD represent fusion with GAL4 activation domain or binding domain, respectively. SD/-LT indicates synthetic drop-out medium lacking leucine and tryptophan. SD/-AHLT indicates media lacking adenine, histidine, leucine, tryptophan and SD/-AHLT + X-α-gal indicates SD/-AHLT plus 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-α-gal). p53_BD and T-antigen_AD are positive controls while Lam_BD and T-antigen_AD are negative controls. ND, 10 -1 , 10 -2 , 10 -3 denote no dilution, 10-fold dilution, 100-fold dilution and 1000-fold dilution respectively, of co-transformed yeast cultures. (D) BiFC assays show subcellular localisation of dimers of ZOS1-15 and its interactors in Nicotiana benthamiana leaf cells. YFP N and YFP C indicate N-terminal and C-terminal regions of YFP in fusion with the mentioned protein. Plasma membrane has been indicated by red arrow and nucleus is indicated by blue arrow. Left panel indicates reconstituted YFP signal at 514 nm. Merged indicate the merger of YFP and bright field and the right column shows the zoomed-in image of the interactions. Scale bar is 50 μm. Pull down and co-immunoprecipitation assays, respectively, confirm (E-F) homodimerisation of ZOS1-15 [GST_ZOS1-15 (44.92 kDa), YFP_ZOS1-15 (46.81 kDa)] and its interaction with (G-H) MED14_1 [GST_MED14_1 (74.51 kDa), YFP_ZOS1-15 (46.81 kDa)]; (I-J) HDT702 [MBP_HDT702 (66.26 kDa), GST_ZOS1-15 (44.92 kDa), YFP_ZOS1-15 (46.81 kDa)]; (K-L) TPL [MBP_OsTPL (74.07 kDa), GST_ZOS1-15 (44.92 kDa), YFP_ZOS1-15 (46.81 kDa)]; (M-N) ONAC024 [GST_ZOS1-15 (44.92 kDa), YFP_ONAC024 (62.54 kDa)]. Input, pull down fractions and the antibody used have been marked. Pull down and Co-IP lane with expected protein bands have been indicated by red arrow. IB denotes immunoblot.

Article Snippet: MBP-fused proteins were purified using MBP resin and the GST-tagged proteins were purified with glutathione agarose beads (G-biosciences ® ; USA).

Techniques: Zinc-Fingers, Activation Assay, Binding Assay, Transformation Assay, Membrane, Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot

Journal: bioRxiv

Article Title: A novel repressor-activator-competitor module comprising C 2 H 2 zinc finger and NAC transcription factors regulates rice grain development

doi: 10.1101/2024.07.15.603577

Figure Lengend Snippet: (A) Pull down assay showing interaction of MBP_ONAC024 (76.91 kDa) with GST_ONAC023 (53.97 kDa). (B) In-planta co-immunoprecipitation assay confirming the interaction of YFP_ONAC024 (62.54kDa) with ONAC023_MYC (39.97kDa). Pull down and Co-IP lanes with expected protein bands have been indicated by red arrows. IB denotes immunoblot. Graphs representing reproductive traits in 023_OE lines compared to WT; (C) average per grain length; (D) average per grain width and (E) average per grain weight (n = 50*3). (F) Transverse sections representing grain chalkiness in wild type (top), and 023_OE grains (5.1 – T3 grains; 36.1 – T4 grains); scale bar = 100µm. (G) Structural analysis of starch granules in WT and 023_OE lines by SEM, scale bar = 10µM; polyhedral starch granules have been marked in yellow and irregular shaped ones are in pink. (H) Estimation of AAC in mature seeds of WT and ONAC023 OE lines. Student’s t-test was used for calculating the significance ( *** = p-value< 0.005).

Article Snippet: MBP-fused proteins were purified using MBP resin and the GST-tagged proteins were purified with glutathione agarose beads (G-biosciences ® ; USA).

Techniques: Pull Down Assay, Co-Immunoprecipitation Assay, Western Blot, Starch

Journal: bioRxiv

Article Title: A novel repressor-activator-competitor module comprising C 2 H 2 zinc finger and NAC transcription factors regulates rice grain development

doi: 10.1101/2024.07.15.603577

Figure Lengend Snippet: Mature grains of 025_SOE (T3 grains) and WT arranged (A) lengthwise and (B) widthwise. Bar, 1 cm. Bar graphs show (C) average per grain length, (D) average per grain width and (E) yield per plant in WT and 025_SOE plants, (n= 50*3). (F) Transverse sections of WT and 025_SOE grains under the stereomicroscope. Scale bars, 1 mm. (G) Morphology profile of compound-type starch granules in the central endosperm sections of 025_SOE mature grains in comparison with WT. The transverse hand-cut sections of grains have been viewed under SEM. The polyhedral starch granules are outlined with yellow dashed lines and irregular granules have been marked in pink. Scale bars, 10µm. Line graph shows (H) AAC in WT and SS1/ ONAC025 transgenic grains and (I) TPC of WT and 025_SOE grains. The data represents two independent transgenic lines with three biological replicates each. The graphs depict average ± SE and statistical significance by one-tailed Student’s t-test ( * p<0.05, ** p<0.01, *** p<0.005). (J) Pull-down assay shows the interaction of MBP_SS1/ ONAC025 (75.8 kDa) with GST_MADS29 (54.86 kDa). The red arrow indicates the expected protein bands in the pull-down lane. IB, immunoblot. (K) Co-immunoprecipitation (Co-IP) assay confirming the interaction between SS1/ ONAC025 (61.43 kDa) and MADS29 (40.86 kDa). Input indicates total protein isolated from N. benthamiana leaves infiltrated with YFP_SS1/ ONAC025 and MADS29_MYC. The red arrow represents immunoprecipitated YFP_SS1/ ONAC025 in the protein mixture. IB denotes immunoblot.

Article Snippet: MBP-fused proteins were purified using MBP resin and the GST-tagged proteins were purified with glutathione agarose beads (G-biosciences ® ; USA).

Techniques: Starch, Comparison, Transgenic Assay, One-tailed Test, Pull Down Assay, Western Blot, Co-Immunoprecipitation Assay, Isolation, Immunoprecipitation

Journal: Journal of Functional Biomaterials

Article Title: Influence of the Application Time of Silane for the Bonding Performance between Feldspar or Lithium Disilicate Ceramics and Luting Resin Composites

doi: 10.3390/jfb14040231

Figure Lengend Snippet: Materials used in the present study.

Article Snippet: Luting resin composite , For MBP Specimen , Calibra translucent , Dentsply DeTrey GmbH Konstanz, Germany , 00041643 00023656.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Micropeptide ASAP encoded by LINC00467 promotes colorectal cancer progression by directly modulating ATP synthase activity

doi: 10.1172/JCI152911

Figure Lengend Snippet: ( A ) Schematic diagram showing the identification of ASAP-interacting proteins using MS analysis following immunoprecipitation. ( B and C ) SFB-ASAP and ATP5A-His ( B ) and ATP5C-His ( C ) were cooverexpressed in HEK293T cells. Co-IP assays were performed to detect the interaction in ASAP-ATP5A ( B ) and ASAP-ATP5C ( C ). ( D ) Co-IP assays showing the endogenous interaction between ASAP and both ATP5A and ATP5C in HCT116 and RKO cells. ( E and F ) In vitro pulldown assays were performed using bacterial purified MBP-ATP5A-His, MBP-ATP5C-His, and GST-ASAP to confirm the direct interaction between ASAP and both ATP5A and ATP5C. ( G ) HEK293T cells transfected with the indicated constructs were used for sequential immunoprecipitation assay with Flag-tag immunomagnetic beads, followed by anti-Myc antibody. Data are representative of 3 independent experiments ( B – G ).

Article Snippet: GST-tagged protein was incubated with MBP-tagged protein in 500 μL of binding buffer (50 mM Tris-HCl, 10% glycerol, 100 mM KCl, 5 mM MgCl 2 , 10 mM β-mercaptoethanol, and 0.1% NP-40, pH 8.0) supplemented with corresponding MBP resin (Sangon Biotech) or GST resin (Sangon Biotech) at 4°C for 3 hours with gentle rotation.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, In Vitro, Purification, Transfection, Construct, FLAG-tag

Journal: The Journal of Clinical Investigation

Article Title: Micropeptide ASAP encoded by LINC00467 promotes colorectal cancer progression by directly modulating ATP synthase activity

doi: 10.1172/JCI152911

Figure Lengend Snippet: ( A and B ) The interaction between ATP5A and ATP5C was detected using Co-IP assays in HCT116 and RKO cells with overexpressed ASAP ( A ) or ASAP KO ( B ). ( C ) In vitro pulldown assay was performed using bacterial purified MBP-ATP5A-His and GST-ATP5C in the presence or absence of ASAP-His to confirm that ASAP directly enhances the interaction between ATP5A and ATP5C. ( D and E ) The activities of respiratory complex V (ATP synthase) were investigated in HCT116 and RKO cells with ASAP overexpressed ( D ) or ASAP KO ( E ). Data are presented as mean value ± SD. n = 6 biologically independent experiments. Independent samples; 2-sided Student’s t test. *** P < 0.001. ( F ) Schematic diagram showing the interaction between ASAP and ATP synthase. The ATP synthase structural model was constructed based on Yoshida et al. ( 67 ). Data are representative of 3 independent experiments ( A – C ).

Article Snippet: GST-tagged protein was incubated with MBP-tagged protein in 500 μL of binding buffer (50 mM Tris-HCl, 10% glycerol, 100 mM KCl, 5 mM MgCl 2 , 10 mM β-mercaptoethanol, and 0.1% NP-40, pH 8.0) supplemented with corresponding MBP resin (Sangon Biotech) or GST resin (Sangon Biotech) at 4°C for 3 hours with gentle rotation.

Techniques: Activity Assay, Co-Immunoprecipitation Assay, In Vitro, Purification, Construct

Journal: The Journal of Clinical Investigation

Article Title: Micropeptide ASAP encoded by LINC00467 promotes colorectal cancer progression by directly modulating ATP synthase activity

doi: 10.1172/JCI152911

Figure Lengend Snippet: ( A ) WT or mutant ASAP was cooverexpressed with SFB-ATP5A in HEK293T cells. Co-IP assays showed that K79A mutation on ASAP abolished the interaction between ASAP and ATP5A. ( B ) WT or mutant ASAP was cooverexpressed with SFB-ATP5C in HEK293T cells. Co-IP assays showed that D65A mutation on ASAP abolished the interaction between ASAP and ATP5C. ( C ) WT or mutant SFB-ATP5A was cooverexpressed with ASAP-His in HEK293T cells. Co-IP assays showed that D454A mutation on ATP5A abolished the interaction between ASAP and ATP5A. ( D ) WT or mutant SFB-ATP5C was cooverexpressed with ASAP-His in HEK293T cells. Co-IP assays showed that K197A mutation on ATP5C abolished the interaction between ASAP and ATP5C. ( E ) The endogenous interaction between ATP5A and ATP5C in HCT116 and RKO cells with ASAP overexpressed or Mut-ASAP was investigated using Co-IP assays. ( F ) The endogenous interaction between ATP5A and ATP5C in HCT116 and RKO cells with ASAP KO or ASAP restored was investigated using Co-IP assays. ( G ) In vitro pulldown assays were performed using bacterial purified MBP-ATP5A-His and GST-ATP5C in the presence of WT ASAP or mutant ASAP. Data are representative of 3 independent experiments ( A – G ).

Article Snippet: GST-tagged protein was incubated with MBP-tagged protein in 500 μL of binding buffer (50 mM Tris-HCl, 10% glycerol, 100 mM KCl, 5 mM MgCl 2 , 10 mM β-mercaptoethanol, and 0.1% NP-40, pH 8.0) supplemented with corresponding MBP resin (Sangon Biotech) or GST resin (Sangon Biotech) at 4°C for 3 hours with gentle rotation.

Techniques: Mutagenesis, Co-Immunoprecipitation Assay, In Vitro, Purification