rabbit anti-gal4-bd (sc-577)  (Santa Cruz Biotechnology)

Journal: PLOS Biology

Article Title: Activation of the Drosophila innate immune system accelerates growth in cooperation with oncogenic Ras

doi: 10.1371/journal.pbio.3003068

Figure Lengend Snippet: Primary antibodies used in this study.

Article Snippet: rabbit polyclonal anti-Gal4 (DBD): sc-577 , Santa Cruz Biotechnology , n/a , 1:1,000.

Techniques:

Journal: bioRxiv

Article Title: Lotus japonicus VIH2 is an inositol pyrophosphate synthase that regulates arbuscular mycorrhiza

doi: 10.1101/2024.12.17.628921

Figure Lengend Snippet: The S. cerevisiae wildtype or vip1 Δ mutant strain was transformed with the episomal pAG426GPD- ccdB empt vector (EV) or with plasmids carrying sequences encoding either a short (LjVIH2 sKD ) or a long (LjVIH2 lKD ) versio of the isolated L. japonicus VIH2 kinase domain fused to a C-terminal V5-tag. Gal4 served as loading control.

Article Snippet: A rabbit anti-Gal4 antibody (Santa Cruz, 1:1000 dilution) followed by a secondary Alexa Fluor TM Plus 800 goat anti-rabbit antibody (Invitrogen, 1:10 000 dilution) was used as loading control.

Techniques: Mutagenesis, Transformation Assay, Plasmid Preparation, Isolation, Control

Journal: Biochemical Journal

Article Title: Comprehensive comparative assessment of the Arabidopsis thaliana MLO2–CALMODULIN2 interaction by various in vitro and in vivo protein–protein interaction assays

doi: 10.1042/BCJ20230255

Figure Lengend Snippet: ( A ) Classical Y2H assay with the pDEST32 (bait vector; Gal4 DNA-binding domain (BD); MLO2 CT , MLO2 CT-LW/RR and empty vector) and pDEST22 (prey vector; Gal4 activation domain (AD); CAM2 and empty vector) vector system in S. cerevisiae strain PJ69-4A. Growth control was performed on SC medium lacking leucine (−L, selection for bait vector) and tryptophan (−T, selection for prey vector). Selection for interaction was performed on SC medium lacking leucine (−L), tryptophan (−T), and histidine (−H, selection for interaction). The assay was repeated twice with similar results. ev, empty vector. ( B ) Classical Y2H assay with the pGBKT7 (bait vector; Gal4 DNA-binding domain (BD); MLO2 CT and MLO2 CT-LW/RR and empty vector) and pGADT7 (prey vector; Gal4 activation domain (AD); CAM2 and empty vector) vector system in S. cerevisiae strain AH109. Growth control was performed on SC medium lacking leucine (−L, selection for bait vector) and tryptophan (−T, selection for prey vector). Selection for interaction was performed on SC medium lacking leucine (−L), tryptophan (−T), and histidine (−H, selection for interaction). The assay was repeated twice with similar results. ev, empty vector. ( C ) Ura3-based yeast SUS with the pMet-GWY-Cub-R-Ura3 (bait vector; MLO2 CT and MLO2 CT-LW/RR ) and pCup-NuI-GWY-Cyc1 (prey vector; CAM2 and GPA1) vector system in S. cerevisiae strain JD53. Growth control was performed on SC medium lacking histidine (−H, selection for bait vector) and tryptophan (−T, selection for prey vector). Selection for interaction was performed on SC medium containing 0.7 g/L 5-FOA (+5-FOA, selection for interaction) and lacking methionine (−M, to allow for full promoter activity of the bait vector). All plates contained 500 µM methionine to reduce background growth due to a strong promoter activity of the bait vector. The assay was repeated twice with similar results. D PLV-based yeast SUS with the pMetOYC (bait vector; MLO2 CT and MLO2 CT-LW/RR ) and pNX32 (prey vector; CAM2) vector system in the S. cerevisiae strain THY.AP4. Growth control was performed on SC medium lacking leucine (−L, selection for bait vector) and tryptophan (−T, selection for prey vector). Selection for interaction was performed either on SC medium lacking leucine (−L), tryptophan (−T) and histidine (−H, selection for interaction) and in the presence of 10 mM 3-aminotriazole (+3-AT) or on SC medium lacking leucine (−L), tryptophan (−T) and adenine (−A, selection for interaction). All plates contained 500 µM methionine to reduce background growth due to a strong promoter activity of the bait vector. The assay was repeated twice with similar results. ev, empty vector. ( E ) Quantification of interaction strength in the PLV-based yeast SUS via a β-galactosidase reporter assay. Yeast cells were harvested from freshly grown cultures (OD 600 = 1) and washed with sterile Z-buffer. Cells were disrupted by three freeze-and-thaw cycles, and the debris was separated by centrifugation. The protein concentration of the supernatant was determined. Aliquots of the supernatant, Z buffer, and the substrate o-nitrophenyl-β- d -galactopyranoside were mixed and incubated at 37°C. The yellowing of the solution was monitored over time and stopped by the addition of Na 2 CO 3 (final concentration 0.33 M). The extinction was measured at 420 nm, and the specific enzyme activity (U/mg) was calculated. Three independent biological replicates were performed and all data points are indicated. An ordinary one-way ANOVA followed by Tukey's multiple comparison testing revealed no statistically significant differences between samples.

Article Snippet: For immunoblot analyses, the following commercially available primary antibodies were used: rabbit α-GST (Cell Signaling Technology, Danvers, MA, U.S.A.; used in 1 : 1000 dilution), mouse α-His (Cell Signaling Technology; used in 1 : 1000 dilution), rat α-HA (Hoffmann-La Roche AG, Basel, Switzerland; used in 1 : 1000 dilution), goat α-luciferase (Sigma–Aldrich, St. Louis, MO, U.S.A.; used in 1 : 1000 dilution), rabbit α-Gal4 BD (Santa Cruz Biotechnology, Dallas, TX, U.S.A.; used in 1 : 1000 dilution), rabbit α-Gal4 AD (Santa Cruz Biotechnology; used in 1 : 1000 dilution), and goat α-biotin-HRP (Cell Signaling Technology; used in 1 : 2000 dilution).

Techniques: Y2H Assay, Plasmid Preparation, Binding Assay, Activation Assay, Selection, Activity Assay, Reporter Assay, Sterility, Centrifugation, Protein Concentration, Incubation, Concentration Assay, Comparison

Journal: Biochemical Journal

Article Title: Comprehensive comparative assessment of the Arabidopsis thaliana MLO2–CALMODULIN2 interaction by various in vitro and in vivo protein–protein interaction assays

doi: 10.1042/BCJ20230255

Figure Lengend Snippet: ( A ) Classical Y2H assay with the pDEST32 (bait vector; Gal4 DNA-binding domain (BD); MLO2 CT , MLO2 CT-LW/RR and empty vector) and pDEST22 (prey vector; Gal4 activation domain (AD); CAM2 and empty vector) vector system in S. cerevisiae strain PJ69-4A. Growth control was performed on SC medium lacking leucine (−L, selection for bait vector) and tryptophan (−T, selection for prey vector). Selection for interaction was performed on SC medium lacking leucine (−L), tryptophan (−T), and histidine (−H, selection for interaction). The assay was repeated twice with similar results. ev, empty vector. ( B ) Classical Y2H assay with the pGBKT7 (bait vector; Gal4 DNA-binding domain (BD); MLO2 CT and MLO2 CT-LW/RR and empty vector) and pGADT7 (prey vector; Gal4 activation domain (AD); CAM2 and empty vector) vector system in S. cerevisiae strain AH109. Growth control was performed on SC medium lacking leucine (−L, selection for bait vector) and tryptophan (−T, selection for prey vector). Selection for interaction was performed on SC medium lacking leucine (−L), tryptophan (−T), and histidine (−H, selection for interaction). The assay was repeated twice with similar results. ev, empty vector. ( C ) Ura3-based yeast SUS with the pMet-GWY-Cub-R-Ura3 (bait vector; MLO2 CT and MLO2 CT-LW/RR ) and pCup-NuI-GWY-Cyc1 (prey vector; CAM2 and GPA1) vector system in S. cerevisiae strain JD53. Growth control was performed on SC medium lacking histidine (−H, selection for bait vector) and tryptophan (−T, selection for prey vector). Selection for interaction was performed on SC medium containing 0.7 g/L 5-FOA (+5-FOA, selection for interaction) and lacking methionine (−M, to allow for full promoter activity of the bait vector). All plates contained 500 µM methionine to reduce background growth due to a strong promoter activity of the bait vector. The assay was repeated twice with similar results. D PLV-based yeast SUS with the pMetOYC (bait vector; MLO2 CT and MLO2 CT-LW/RR ) and pNX32 (prey vector; CAM2) vector system in the S. cerevisiae strain THY.AP4. Growth control was performed on SC medium lacking leucine (−L, selection for bait vector) and tryptophan (−T, selection for prey vector). Selection for interaction was performed either on SC medium lacking leucine (−L), tryptophan (−T) and histidine (−H, selection for interaction) and in the presence of 10 mM 3-aminotriazole (+3-AT) or on SC medium lacking leucine (−L), tryptophan (−T) and adenine (−A, selection for interaction). All plates contained 500 µM methionine to reduce background growth due to a strong promoter activity of the bait vector. The assay was repeated twice with similar results. ev, empty vector. ( E ) Quantification of interaction strength in the PLV-based yeast SUS via a β-galactosidase reporter assay. Yeast cells were harvested from freshly grown cultures (OD 600 = 1) and washed with sterile Z-buffer. Cells were disrupted by three freeze-and-thaw cycles, and the debris was separated by centrifugation. The protein concentration of the supernatant was determined. Aliquots of the supernatant, Z buffer, and the substrate o-nitrophenyl-β- d -galactopyranoside were mixed and incubated at 37°C. The yellowing of the solution was monitored over time and stopped by the addition of Na 2 CO 3 (final concentration 0.33 M). The extinction was measured at 420 nm, and the specific enzyme activity (U/mg) was calculated. Three independent biological replicates were performed and all data points are indicated. An ordinary one-way ANOVA followed by Tukey's multiple comparison testing revealed no statistically significant differences between samples.

Article Snippet: For immunoblot analyses, the following commercially available primary antibodies were used: rabbit α-GST (Cell Signaling Technology, Danvers, MA, U.S.A.; used in 1 : 1000 dilution), mouse α-His (Cell Signaling Technology; used in 1 : 1000 dilution), rat α-HA (Hoffmann-La Roche AG, Basel, Switzerland; used in 1 : 1000 dilution), goat α-luciferase (Sigma–Aldrich, St. Louis, MO, U.S.A.; used in 1 : 1000 dilution), rabbit α-Gal4 BD (Santa Cruz Biotechnology, Dallas, TX, U.S.A.; used in 1 : 1000 dilution), rabbit α-Gal4 AD (Santa Cruz Biotechnology; used in 1 : 1000 dilution), and goat α-biotin-HRP (Cell Signaling Technology; used in 1 : 2000 dilution).

Techniques: Y2H Assay, Plasmid Preparation, Binding Assay, Activation Assay, Control, Selection, Activity Assay, Reporter Assay, Sterility, Centrifugation, Protein Concentration, Incubation, Concentration Assay, Comparison