Journal: bioRxiv

Article Title: Linking sugar sensing to immunity in plants via O-glycosylation

doi: 10.64898/2026.05.04.722566

Figure Lengend Snippet: (A) MKK5-GFP was affinity purified from 7-day-old WT but not spy-4 seedlings using Aleuria Aurantia Lectin (AAL) immobilized on agarose beads. Coomassie Brilliant Blue staining (CBB) of the membrane shows the loading control. (B) Bio-layer interferometry (BLI) assay showing binding kinetics between SPY and MKK4. 6xHis-SUMO-3TPR-SPY was immobilized to anti-His sensors and dipped into indicated concentrations of GST-MKK4 protein. The dissociation constant (K d ) was calculated by steady-state fitting of GST-MKK4 concentration against the response unit. (C) Co-immunoprecipitation shows in vivo interaction between SPY and MKK5. YFP or SPY-YFP was transiently co-expressed with MKK5-myc in N. benthamiana leaves, immunoprecipitated by anti-GFP antibody, and immunoblotted with anti-GFP or anti-myc antibody. (D) in vitro affinity purification (pull-down) assay shows direct interaction between SPY/SEC and MKK4/5. Recombinant GST, GST-MKK4, and GST-MKK5 were incubated with 6xHis-tagged SPY, SEC, and PCK1, affinity-purified by Glutathione-agarose beads, and immunoblotted using anti-His and anti-GST antibodies. (E) Higher energy collisional dissociation (HCD) mass spectrum shows O-fucosylation on the activation loop peptide of MKK4 spanning amino acids 220-242, after in vitro O-fucosylation by SPY.

Article Snippet: In brief, bait proteins (final concentration 20 μg/mL) were immobilized onto anti-His (Gator bio #160009) or anti-GST biosensors (Gator bio, #160042) using a Gator-plus instrument (Gator Bio).

Techniques: Affinity Purification, Staining, Membrane, Control, Binding Assay, Concentration Assay, Immunoprecipitation, In Vivo, In Vitro, Pull Down Assay, Recombinant, Incubation, Activation Assay

Journal: bioRxiv

Article Title: Linking sugar sensing to immunity in plants via O-glycosylation

doi: 10.64898/2026.05.04.722566

Figure Lengend Snippet: (A) Bio-layer interferometry (BLI) showing binding kinetics between SPY and MKK5 . 6xHis-SUMO-3TPR-SPY was immobilized to anti-His sensors and dipped into indicated concentrations of GST-MKK5 proteins. The dissociation constant (K d ) was calculated by steady-state fitting of GST-MKK5 concentration against the response unit. (B) BLI assay showing interaction between O-fucosylated GST-MKK5 protein with AAL. GST-MKK5 was incubated with SPY in vitro, with or without GDP-fucose (GF) and SOFTI, loaded onto anti-GST biosensors, and analyzed for binding to AAL.

Article Snippet: In brief, bait proteins (final concentration 20 μg/mL) were immobilized onto anti-His (Gator bio #160009) or anti-GST biosensors (Gator bio, #160042) using a Gator-plus instrument (Gator Bio).

Techniques: Binding Assay, Concentration Assay, Incubation, In Vitro

Journal: Biotechnology Reports

Article Title: Comparative analysis of anti-MICA scFv affinities: Insights from three label-free biophysical methods and biological validation

doi: 10.1016/j.btre.2026.e00955

Figure Lengend Snippet: Characterization of Recombinant Proteins: MICA and anti-MICA scFvs. (A) Molecular model, shown as a ribbon representation, of the variable fragment of the anti-MICA scFvs. The framework is displayed in white, the light chain CDRs are shown in cyan, and the heavy chain CDRs are shown in yellow. The residues with mutations are shown as magenta spheres [residues 32 (CDR L1), 164 (CDR H1), and 188/190 (CDR H2)]. (B) Schematic diagram of the scFv gene. The modified pET-15b vector was used for the expression of the WT and Beta mutant scFvs, each carrying four mutations: I32Y in CDR1 of the VL, and S164F, P188W, and G190W in CDR1, CDR2, and CDR2 of the VH, respectively. Recombinant proteins were expressed in E. coli BL21(DE3). (C) SDS-PAGE analysis showing the purity of recombinant proteins: WT scFv, Beta mutant scFv, and MICA. Proteins were resolved on a 12% acrylamide gel under reducing conditions. SDS-PAGE results show the soluble fraction (SF), unbound protein (UBP), elution of purified scFv (E), renatured proteins (R) and inclusion bodies (IB). MW, molecular weight. (D-E) Western blot analysis confirming the identity of scFvs and MICA using an anti-HisTag antibody. For the identification of the WT and Beta mutant scFvs, Anti-6xHis Epitope Tag mouse monoclonal antibody conjugated with peroxidase (200-303-382) was used at a dilution of 1:1000. For the identification of MICA, a biotinylated Anti-MICA antibody (BAMO3 (BAFI300, BamOmaB)) and Streptavidin were used at a dilution of 1:2000. A total of 2 μg of purified protein was loaded. The negative control (Ctrl -) for MICA detection was WT scFv and MICA protein was used for scFv detection. Original gel is presented in Fig. S1, Supplementary information.

Article Snippet: The identity of MICA and scFvs proteins was confirmed by western blot using a HRP-conjugated anti-His tag monoclonal antibody (200-303-382, Rockland, USA).

Techniques: Recombinant, Modification, Plasmid Preparation, Expressing, Mutagenesis, SDS Page, Acrylamide Gel Assay, Purification, Molecular Weight, Western Blot, Negative Control

Journal: Transboundary and Emerging Diseases

Article Title: Toxoplasma gondii KCR is a Noncanonical Modulator of CSF2 Signaling that Targets the CSF2Rα–JAK2/STAT5 Axis

doi: 10.1155/tbed/8426765

Figure Lengend Snippet: Protein interactions and eukaryotic protein acquisition. (A) Schematic overview of the screening strategy and identification of T. gondii KCR. (B) Co‐immunoprecipitation identification of the interaction between KCR and murine CSF2Rα input: cell lysates from HEK 293T cells co‐transfected with pcDNA3.1‐KCR and pCAGGS‐CSF2R for 24 h; IP: KCR, CSF2α or IgG: immunoprecipitation was performed using Flag‐tag mouse mAb, His‐tag mouse mAb or mouse IgG; IB: KCR or CSF2α: immunoblot analysis was performed using Flag‐tag rabbit mAb or His‐tag rabbit pAb. (C) Acquisition of KCR eukaryotic protein. Lane M: standard molecular marker for protein; lane 1: cell lysates from HEK 293T cells transfected with pcDNA3.1‐KCR for 24 h; lane 2: purified KCR eukaryotic protein. (D) Western blot analysis of KCR M: standard molecular marker for protein; lane 3: his‐tag in purified KCR was identified by His‐tag mouse mAb.

Article Snippet: Flag‐tag mouse monoclonal antibody (mAb) (#M20008), His‐tag mouse mAb (# M20001 ), and mouse IgG (#B30010M) were purchased from Abmart Biotech, Inc. (Shanghai, China).

Techniques: Immunoprecipitation, Transfection, FLAG-tag, Western Blot, Marker, Purification