Journal: iScience

Article Title: Borrelia-specific antibody profiles and complement deposition in joint fluid distinguish antibiotic-refractory from -responsive Lyme arthritis

doi: 10.1016/j.isci.2024.108804

Figure Lengend Snippet: Compartment-specific responses differ across antibiotic responsiveness The polar plots show the mean percentile rank for each antigen for sambucus nigra (SNA) lectin binding, Ricinus communis agglutinin 1 (RCA) lectin binding, and antibody-dependent complement deposition (ADCD) measurements for (A) refractory joint fluid, (B) refractory serum, (C) responsive joint fluid, and (D) responsive serum. The antigen key indicates the corresponding antigen for each colored slice of the polar plot.

Article Snippet: Sambucus Nigra Lectin (SNA, EBL), Fluorescein , Vectorlabs , Cat# FL-1301-2.

Techniques: Binding Assay

Journal: iScience

Article Title: Borrelia-specific antibody profiles and complement deposition in joint fluid distinguish antibiotic-refractory from -responsive Lyme arthritis

doi: 10.1016/j.isci.2024.108804

Figure Lengend Snippet:

Article Snippet: Sambucus Nigra Lectin (SNA, EBL), Fluorescein , Vectorlabs , Cat# FL-1301-2.

Techniques: Recombinant, Produced, Generated, Software, Luminex

Journal: bioRxiv

Article Title: Sialylated flagellin from Pseudomonas aeruginosa modulates the process of dendritic cell maturation through Siglec-9 mediated suppression of TLR5 signaling

doi: 10.1101/2025.03.21.644494

Figure Lengend Snippet: Evaluation of the sialylation status of flagellin isolated from PA +Sia (A) The sialic acid specific – Sambucus nigra (SNA) lectin conjugated to agarose beads was used to isolate sialoglycoproteins from PA +Sia lysate (100 µg) by a lectin-based pulldown assay. Highly sialylated bovine salivary mucin (BSM) was used as a positive control. In the case of PA +Sia lysate, the lectin bound sialylated proteins were resolved in 10% SDS-PAGE and visualized by Coomassie Blue staining. Staining revealed several fine bands along with a prominent band near 47 kDa and two bands near 34, 37 kDa. The presence of similar bands in the BSM control indicated that these 34, 37 kDa bands come from the SNA-agarose beads. The band near 47 kDa was excised and processed for in gel trypsin digestion. The protein band was reduced, alkylated then digested by trypsin and subjected to MALDI TOF MS/MS. The generated spectra were searched against a eubacterial protein database by the MASCOT search engine utilizing the Basic Local Alignment Tool (BLAST) of ABI GPS Explorer software, version 3.6. Through peptide mass fingerprinting, the protein band was identified as flagellin. The MS/MS spectra with each tryptic fragments denoted by respective m/z values and the BLAST search results are shown. The peptide fragments within the flagellin sequence which were detected via MALDI TOF MS/MS have been marked with red colour. (B) The isolated, purified Fla +Sia protein (2 μg) resolved to a single band in a 10% SDS-PAGE gel. This single band was carefully excised and subjected to in gel trypsin digestion as before. The peptide digest was analyzed via ESI MS/MS mode by the LTQ Orbitrap mass spectrometer. The generated spectra was searched against the eubacterial protein database compiled from Uniprot using the Sequest HT Proteome Discoverer 1.4 software (Thermo Fisher Scientific) utilizing the MASCOT search engine. All peptide fragments among the flagellin sequence which have been identified in the tryptic digest have been denoted with red colour. (C) Purified Fla +Sia (25 μg) was passively rehydrated into pH 4–7 IPG strips and resolved via isoelectric focussing. The resolved strips were placed over the resolving gel in a10% SDS-PAGE gel and fixed using melted agarose. The protein sample was resolved in the second dimension through electrophoresis. The 2D gels were stained using Coomassie Blue to reveal that the single band protein had split into seven isoforms . (D) The sialic acid content of Fla +Sia /Fla -Sia (500 µg) were estimated and compared using the fluorimetric acetyl acetone method following Shukla et al (1982). Following the acetyl-acetone reaction, sialic acids released from flagellin proteins formed a fluorigenic product which were quantified via a fluorimeter (Ex-410nm, Em-510nm). The amount of sialic acids present in each sample was calculated by comparing with a prepared standard dilution of synthetic N-acetyl Neuraminic acid. Data from three independent experiments were used to prepare the bar diagram shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (E) Fla +Sia /Fla -Sia (5μg) resolved via SDS-PAGE was transferred onto PVDF blots via wet transfer method. These blots were probed with biotinylated sialic acid binding lectins – Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA II). The blots were washed and incubated with secondary avidin-HRP and the extent of lectin binding was determined by developing the blot using chemiluminescent substrates. The Ponceau S stained blots are provided to account for equal protein loading. Densitometric data from three independent experiments were used to prepare the bar diagram shown here. Representative blots from one experiment are shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (F) Fla +Sia was used to coat a 96 well ELISA plate. A panel of Siglec-Fc chimera recombinant proteins (Siglec-3,4,5,6,7,9 and 10-Fc) was diluted and incubated in these Fla +Sia coated wells. The wells were then washed and incubated with anti-human IgG Fc-specific peroxidase secondary antibodies. The extent of Siglec-Fc remaining bound to the Fla +Sia coated wells were detected using TMB substrate. The colour which was generated from the bound secondary antibodies was measured as absorbance at 450nm in a plate reader. Data from three independent experiments were used to prepare the bar diagram shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (G) As before, Fla +Sia /Fla -Sia (5μg) were resolved in SDS-PAGE, transferred to blots and probed using the same Siglec-Fc panel. The blots were washed and then incubated with secondary anti-human IgG Fc region specific peroxidase antibodies. The extent of Siglec binding was directly visualized by developing the blots in a chemidoc using a chemiluminescent substrate. As before, Ponceau S stained blots are supplied to account for equal loading. One representative blot from three independent experiments performed is shown here.

Article Snippet: Vectashield mounting medium, biotinylated Sambucus nigra agglutinin (SNA) (#B-1305-2), biotinylated Maackia amurensis agglutinin (MALII) (#B-1265-1), agarose-SNA lectin beads (AL-1303-2) and biotinylated Peanut agglutinin (B-1075-5) were obtained from Vector Laboratories (USA).

Techniques: Isolation, Positive Control, SDS Page, Staining, Control, Tandem Mass Spectroscopy, Generated, Software, Peptide Mass Fingerprinting, Sequencing, Purification, Mass Spectrometry, Electrophoresis, Binding Assay, Incubation, Avidin-Biotin Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: bioRxiv

Article Title: Sialylated flagellin from Pseudomonas aeruginosa modulates the process of dendritic cell maturation through Siglec-9 mediated suppression of TLR5 signaling

doi: 10.1101/2025.03.21.644494

Figure Lengend Snippet: (A) Purified Fla +Sia or desialylated Fla +Sia (ds-Fla +Sia ) (20 μg) were passively rehydrated into pH 3-10 IPG strips. The proteins were resolved by isoelectric focusing following the manufacturer’s instructions. These strips were stained by Coomassie blue and then destained before being dried. IPG strips were observed to detect any changes in protein resolution upon sialidase treatment. Representative strips selected from the two independent experiments performed are shown here. (B) As before, Fla +Sia /ds-Fla +Sia (5μg) were resolved in SDS-PAGE, transferred on to PVDF blots and probed with biotinylated sialic acid binding lectins – SNA, MAA II ((5 μg/mL). The blots were washed and then incubated with avidin-HRP. The extent of lectin binding was determined by developing the blots in a chemidoc using a chemiluminescent substrate. Additionally, the galactose specific lectin – peanut agglutinin (PNA) was also used (10 μg/mL). Upon sialidase treatment, the removal of sialic acid residues at the glycan termini exposes the underlying galactose residues. PNA recognizes and binds these exposed galactose residues indicating the successful removal of sialic acids. Densitometric data from three independent experiments were used to calculate the mean band intensities which are shown in these graphs representing the binding studies. One representative blot per experiment is shown here. Significance represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (C) In a same set up, Fla +Sia /ds-Fla +Sia blots were probed with Siglec-9-Fc chimera proteins. The blots were washed and then incubated with anti-human IgG Fc specific antibodies. Any change in Siglec-9 interaction with flagellin was detected by developing the blot in a chemidoc. As previously described, Ponceau S stained blots are shown to account for equal protein loading across lanes. Densitometric data from three independent experiments were used to calculate the mean band intensities which are shown in these graphs representing the binding studies. One representative blot per experiment is shown here. Significance represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Article Snippet: Vectashield mounting medium, biotinylated Sambucus nigra agglutinin (SNA) (#B-1305-2), biotinylated Maackia amurensis agglutinin (MALII) (#B-1265-1), agarose-SNA lectin beads (AL-1303-2) and biotinylated Peanut agglutinin (B-1075-5) were obtained from Vector Laboratories (USA).

Techniques: Purification, Staining, SDS Page, Binding Assay, Incubation, Avidin-Biotin Assay

Journal: Current Genetics

Article Title: Histone 3 lysine 4 monomethylation supports activation of transcription in S. cerevisiae during nutrient stress

doi: 10.1007/s00294-022-01226-2

Figure Lengend Snippet: Location and methylation activity of Set1 mutants. A Cryo-EM structure of S. cerevisiae Set1 in the COMPASS complex (Qu et al. ). COMPASS proteins, Bre2 (purple), Sdc1 (orange), Set1 (yellow), Spp1 (magenta), Swd1 (blue), and Swd3 (green) are shown (Krogan et al. ; Miller et al. ; Nagy et al. ; Roguev et al. ; Takahashi et al. ). The C-terminal tail of Swd1 weaves within the complex to provide a central scaffold for assembly of COMPASS (Qu et al. ; Takahashi et al. ). The Swd2 protein (not shown) connects Swd1 and Swd3 to the N terminus of Set1 (Wang et al. ) and interacts with the CTD of RNA Pol II allowing COMPASS to move along DNA sequences during Pol II transcription (Bae et al. ). Inset, substitution of three amino acid (G951, Y967, and R1013) in or near the Set1 active site generated set1 mutants with different methylation abilities (Williamson et al. ). B Representative Western blot measuring in vivo steady-state levels of H3K4me1 (α-me1), H3K4me2 (α-me2), and H3K4me3 (α-me3) in whole cell extracts from the yeast strains indicated above the blot image ( n = 3). The level of total histone H3 (α-H3) was used to normalize the amount of extract loaded in each lane. Numbers indicate the normalized levels of H3K4me1/2/3 detected in the mutants relative to the wild-type SET1 + strain. Bands labeled 0 (no signal) or > 0.05% (low signal, yet visible by eye) were not detected above background by the Imagequant TL 8.1 image detection software. The samples shown were separated on the same gel and analyzed on the same membranes. Samples between the SET1 + and set1-G951A samples were cropped out of the figure. C Summary of the H3K4 methylation activity in wild-type SET1 and set1 mutants, a filled circle indicates methylation is detectable above background; open circle, methylation is not detected by Imagequant TL 8.1 image detection software (see “ ”)

Article Snippet: The following antibodies were used; α-H3, Abcam ab1791 (4 µg/IP, lot #s: GR3297884-1, GR3297878-1, GR3356864-1 and GR3366670-1), α-H3K4me3, Epicypher 13-0041 (2 µg/IP, lot #s: 20083002-42 and 20218003-49), α-H3K4me2, Epicypher 13-0027 (4 µg/IP lot #: 20252002-04), α-H3K4me1, Epicypher 13-0040 (2 µg/IP lot #s: 19338001-42 and 20178005-44).

Techniques: Methylation, Activity Assay, Cryo-EM Sample Prep, Generated, Western Blot, In Vivo, Labeling, Software

Journal: Current Genetics

Article Title: Histone 3 lysine 4 monomethylation supports activation of transcription in S. cerevisiae during nutrient stress

doi: 10.1007/s00294-022-01226-2

Figure Lengend Snippet: Growth of mutants lacking Set1 methylation activity is sensitive to histidine starvation induced by 3AT. Five-fold serial dilutions of yeast cultures expressing wild-type SET1 + or set1 mutant alleles were spotted onto A SC-complete agar with or without 10 mM 3AT and B SC-His agar with or without 10 mM 3AT. Allele, relevant genotype. Activity, H3K4 methylation activity determined by Western blots (Fig. B); WT, H3K4me1/2/3 detected; Null, no H3K4me1/2/3 detected; mono, H3K4me1 detected with low or no H3K4me2/3; mono/di, H3K4me1/2 detected with no H3K4me3 (Fig. B). Dilution of the cultures, right triangle at the top of each column. Plates were incubated at 30 °C for 42 h prior to imaging. All cultures shown in the figure were grown on the same plate. The images were cut to remove a strain not being considered here

Article Snippet: The following antibodies were used; α-H3, Abcam ab1791 (4 µg/IP, lot #s: GR3297884-1, GR3297878-1, GR3356864-1 and GR3366670-1), α-H3K4me3, Epicypher 13-0041 (2 µg/IP, lot #s: 20083002-42 and 20218003-49), α-H3K4me2, Epicypher 13-0027 (4 µg/IP lot #: 20252002-04), α-H3K4me1, Epicypher 13-0040 (2 µg/IP lot #s: 19338001-42 and 20178005-44).

Techniques: Methylation, Activity Assay, Expressing, Mutagenesis, Western Blot, Incubation, Imaging

Journal: Current Genetics

Article Title: Histone 3 lysine 4 monomethylation supports activation of transcription in S. cerevisiae during nutrient stress

doi: 10.1007/s00294-022-01226-2

Figure Lengend Snippet: Distribution of H3K4me marks at three positions of the HIS3 gene. A Schematic showing the regions of HIS3 gene (promoter, 5′ ORF and 3′ ORF) evaluated by qPCR after ChIP for H3K4me1, H3K4me2 and H3K4me3. B Key to graph. C Association of H3K4me at the HIS3 promoter, 5′ ORF and 3′ ORF in yeast cultures grown in SC-His. D Association of H3K4me at the HIS3 promoter, 5′ ORF and 3′ ORF in yeast cultures grown in SC-HIS + 10 mM 3AT. See Supplementary Table 3 for statistical analysis; error bars, (± SEM, n = 4)

Article Snippet: The following antibodies were used; α-H3, Abcam ab1791 (4 µg/IP, lot #s: GR3297884-1, GR3297878-1, GR3356864-1 and GR3366670-1), α-H3K4me3, Epicypher 13-0041 (2 µg/IP, lot #s: 20083002-42 and 20218003-49), α-H3K4me2, Epicypher 13-0027 (4 µg/IP lot #: 20252002-04), α-H3K4me1, Epicypher 13-0040 (2 µg/IP lot #s: 19338001-42 and 20178005-44).

Techniques:

Journal: bioRxiv

Article Title: Sialylated flagellin from Pseudomonas aeruginosa modulates the process of dendritic cell maturation through Siglec-9 mediated suppression of TLR5 signaling

doi: 10.1101/2025.03.21.644494

Figure Lengend Snippet: Evaluation of the sialylation status of flagellin isolated from PA +Sia (A) The sialic acid specific – Sambucus nigra (SNA) lectin conjugated to agarose beads was used to isolate sialoglycoproteins from PA +Sia lysate (100 µg) by a lectin-based pulldown assay. Highly sialylated bovine salivary mucin (BSM) was used as a positive control. In the case of PA +Sia lysate, the lectin bound sialylated proteins were resolved in 10% SDS-PAGE and visualized by Coomassie Blue staining. Staining revealed several fine bands along with a prominent band near 47 kDa and two bands near 34, 37 kDa. The presence of similar bands in the BSM control indicated that these 34, 37 kDa bands come from the SNA-agarose beads. The band near 47 kDa was excised and processed for in gel trypsin digestion. The protein band was reduced, alkylated then digested by trypsin and subjected to MALDI TOF MS/MS. The generated spectra were searched against a eubacterial protein database by the MASCOT search engine utilizing the Basic Local Alignment Tool (BLAST) of ABI GPS Explorer software, version 3.6. Through peptide mass fingerprinting, the protein band was identified as flagellin. The MS/MS spectra with each tryptic fragments denoted by respective m/z values and the BLAST search results are shown. The peptide fragments within the flagellin sequence which were detected via MALDI TOF MS/MS have been marked with red colour. (B) The isolated, purified Fla +Sia protein (2 μg) resolved to a single band in a 10% SDS-PAGE gel. This single band was carefully excised and subjected to in gel trypsin digestion as before. The peptide digest was analyzed via ESI MS/MS mode by the LTQ Orbitrap mass spectrometer. The generated spectra was searched against the eubacterial protein database compiled from Uniprot using the Sequest HT Proteome Discoverer 1.4 software (Thermo Fisher Scientific) utilizing the MASCOT search engine. All peptide fragments among the flagellin sequence which have been identified in the tryptic digest have been denoted with red colour. (C) Purified Fla +Sia (25 μg) was passively rehydrated into pH 4–7 IPG strips and resolved via isoelectric focussing. The resolved strips were placed over the resolving gel in a10% SDS-PAGE gel and fixed using melted agarose. The protein sample was resolved in the second dimension through electrophoresis. The 2D gels were stained using Coomassie Blue to reveal that the single band protein had split into seven isoforms . (D) The sialic acid content of Fla +Sia /Fla -Sia (500 µg) were estimated and compared using the fluorimetric acetyl acetone method following Shukla et al (1982). Following the acetyl-acetone reaction, sialic acids released from flagellin proteins formed a fluorigenic product which were quantified via a fluorimeter (Ex-410nm, Em-510nm). The amount of sialic acids present in each sample was calculated by comparing with a prepared standard dilution of synthetic N-acetyl Neuraminic acid. Data from three independent experiments were used to prepare the bar diagram shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (E) Fla +Sia /Fla -Sia (5μg) resolved via SDS-PAGE was transferred onto PVDF blots via wet transfer method. These blots were probed with biotinylated sialic acid binding lectins – Sambucus nigra agglutinin (SNA) and Maackia amurensis agglutinin (MAA II). The blots were washed and incubated with secondary avidin-HRP and the extent of lectin binding was determined by developing the blot using chemiluminescent substrates. The Ponceau S stained blots are provided to account for equal protein loading. Densitometric data from three independent experiments were used to prepare the bar diagram shown here. Representative blots from one experiment are shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (F) Fla +Sia was used to coat a 96 well ELISA plate. A panel of Siglec-Fc chimera recombinant proteins (Siglec-3,4,5,6,7,9 and 10-Fc) was diluted and incubated in these Fla +Sia coated wells. The wells were then washed and incubated with anti-human IgG Fc-specific peroxidase secondary antibodies. The extent of Siglec-Fc remaining bound to the Fla +Sia coated wells were detected using TMB substrate. The colour which was generated from the bound secondary antibodies was measured as absorbance at 450nm in a plate reader. Data from three independent experiments were used to prepare the bar diagram shown here. Significance is represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (G) As before, Fla +Sia /Fla -Sia (5μg) were resolved in SDS-PAGE, transferred to blots and probed using the same Siglec-Fc panel. The blots were washed and then incubated with secondary anti-human IgG Fc region specific peroxidase antibodies. The extent of Siglec binding was directly visualized by developing the blots in a chemidoc using a chemiluminescent substrate. As before, Ponceau S stained blots are supplied to account for equal loading. One representative blot from three independent experiments performed is shown here.

Article Snippet: Vectashield mounting medium, biotinylated Sambucus nigra agglutinin (SNA) (#B-1305-2), biotinylated Maackia amurensis agglutinin (MALII) (#B-1265-1), agarose-SNA lectin beads (AL-1303-2) and biotinylated Peanut agglutinin (B-1075-5) were obtained from Vector Laboratories (USA).

Techniques: Isolation, Positive Control, SDS Page, Staining, Control, Tandem Mass Spectroscopy, Generated, Software, Peptide Mass Fingerprinting, Sequencing, Purification, Mass Spectrometry, Electrophoresis, Binding Assay, Incubation, Avidin-Biotin Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: bioRxiv

Article Title: Sialylated flagellin from Pseudomonas aeruginosa modulates the process of dendritic cell maturation through Siglec-9 mediated suppression of TLR5 signaling

doi: 10.1101/2025.03.21.644494

Figure Lengend Snippet: (A) Purified Fla +Sia or desialylated Fla +Sia (ds-Fla +Sia ) (20 μg) were passively rehydrated into pH 3-10 IPG strips. The proteins were resolved by isoelectric focusing following the manufacturer’s instructions. These strips were stained by Coomassie blue and then destained before being dried. IPG strips were observed to detect any changes in protein resolution upon sialidase treatment. Representative strips selected from the two independent experiments performed are shown here. (B) As before, Fla +Sia /ds-Fla +Sia (5μg) were resolved in SDS-PAGE, transferred on to PVDF blots and probed with biotinylated sialic acid binding lectins – SNA, MAA II ((5 μg/mL). The blots were washed and then incubated with avidin-HRP. The extent of lectin binding was determined by developing the blots in a chemidoc using a chemiluminescent substrate. Additionally, the galactose specific lectin – peanut agglutinin (PNA) was also used (10 μg/mL). Upon sialidase treatment, the removal of sialic acid residues at the glycan termini exposes the underlying galactose residues. PNA recognizes and binds these exposed galactose residues indicating the successful removal of sialic acids. Densitometric data from three independent experiments were used to calculate the mean band intensities which are shown in these graphs representing the binding studies. One representative blot per experiment is shown here. Significance represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. (C) In a same set up, Fla +Sia /ds-Fla +Sia blots were probed with Siglec-9-Fc chimera proteins. The blots were washed and then incubated with anti-human IgG Fc specific antibodies. Any change in Siglec-9 interaction with flagellin was detected by developing the blot in a chemidoc. As previously described, Ponceau S stained blots are shown to account for equal protein loading across lanes. Densitometric data from three independent experiments were used to calculate the mean band intensities which are shown in these graphs representing the binding studies. One representative blot per experiment is shown here. Significance represented by *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Article Snippet: Vectashield mounting medium, biotinylated Sambucus nigra agglutinin (SNA) (#B-1305-2), biotinylated Maackia amurensis agglutinin (MALII) (#B-1265-1), agarose-SNA lectin beads (AL-1303-2) and biotinylated Peanut agglutinin (B-1075-5) were obtained from Vector Laboratories (USA).

Techniques: Purification, Staining, SDS Page, Binding Assay, Incubation, Avidin-Biotin Assay

Journal: eLife

Article Title: MS-275, a class 1 histone deacetylase inhibitor augments glucagon-like peptide-1 receptor agonism to improve glycemic control and reduce obesity in diet-induced obese mice

doi: 10.7554/eLife.52212

Figure Lengend Snippet:

Article Snippet: Antibody , SNAP25 (mouse monoclonal) , Santa Cruz , Sc-376713 , Dilution (1:500).

Techniques: Electrofusion, Derivative Assay, Transfection, Construct, Sequencing, Plasmid Preparation, Recombinant, Synthesized, Reporter Gene Assay, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, cDNA Synthesis, Isolation, Software

Journal: eLife

Article Title: Expression of WIPI2B counteracts age-related decline in autophagosome biogenesis in neurons

doi: 10.7554/eLife.44219

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , SNAP-Cell 647-SiR , New England Biolabs , Cat # S9102S , .

Techniques: Transfection, Construct, Generated, Sequencing, Software

Journal: Current Genetics

Article Title: Histone 3 lysine 4 monomethylation supports activation of transcription in S. cerevisiae during nutrient stress

doi: 10.1007/s00294-022-01226-2

Figure Lengend Snippet: Location and methylation activity of Set1 mutants. A Cryo-EM structure of S. cerevisiae Set1 in the COMPASS complex (Qu et al. ). COMPASS proteins, Bre2 (purple), Sdc1 (orange), Set1 (yellow), Spp1 (magenta), Swd1 (blue), and Swd3 (green) are shown (Krogan et al. ; Miller et al. ; Nagy et al. ; Roguev et al. ; Takahashi et al. ). The C-terminal tail of Swd1 weaves within the complex to provide a central scaffold for assembly of COMPASS (Qu et al. ; Takahashi et al. ). The Swd2 protein (not shown) connects Swd1 and Swd3 to the N terminus of Set1 (Wang et al. ) and interacts with the CTD of RNA Pol II allowing COMPASS to move along DNA sequences during Pol II transcription (Bae et al. ). Inset, substitution of three amino acid (G951, Y967, and R1013) in or near the Set1 active site generated set1 mutants with different methylation abilities (Williamson et al. ). B Representative Western blot measuring in vivo steady-state levels of H3K4me1 (α-me1), H3K4me2 (α-me2), and H3K4me3 (α-me3) in whole cell extracts from the yeast strains indicated above the blot image ( n = 3). The level of total histone H3 (α-H3) was used to normalize the amount of extract loaded in each lane. Numbers indicate the normalized levels of H3K4me1/2/3 detected in the mutants relative to the wild-type SET1 + strain. Bands labeled 0 (no signal) or > 0.05% (low signal, yet visible by eye) were not detected above background by the Imagequant TL 8.1 image detection software. The samples shown were separated on the same gel and analyzed on the same membranes. Samples between the SET1 + and set1-G951A samples were cropped out of the figure. C Summary of the H3K4 methylation activity in wild-type SET1 and set1 mutants, a filled circle indicates methylation is detectable above background; open circle, methylation is not detected by Imagequant TL 8.1 image detection software (see “ ”)

Article Snippet: The following antibodies were used; α-H3, Abcam ab1791 (4 µg/IP, lot #s: GR3297884-1, GR3297878-1, GR3356864-1 and GR3366670-1), α-H3K4me3, Epicypher 13-0041 (2 µg/IP, lot #s: 20083002-42 and 20218003-49), α-H3K4me2, Epicypher 13-0027 (4 µg/IP lot #: 20252002-04), α-H3K4me1, Epicypher 13-0040 (2 µg/IP lot #s: 19338001-42 and 20178005-44).

Techniques: Methylation, Activity Assay, Cryo-EM Sample Prep, Generated, Western Blot, In Vivo, Labeling, Software

Journal: Current Genetics

Article Title: Histone 3 lysine 4 monomethylation supports activation of transcription in S. cerevisiae during nutrient stress

doi: 10.1007/s00294-022-01226-2

Figure Lengend Snippet: Growth of mutants lacking Set1 methylation activity is sensitive to histidine starvation induced by 3AT. Five-fold serial dilutions of yeast cultures expressing wild-type SET1 + or set1 mutant alleles were spotted onto A SC-complete agar with or without 10 mM 3AT and B SC-His agar with or without 10 mM 3AT. Allele, relevant genotype. Activity, H3K4 methylation activity determined by Western blots (Fig. B); WT, H3K4me1/2/3 detected; Null, no H3K4me1/2/3 detected; mono, H3K4me1 detected with low or no H3K4me2/3; mono/di, H3K4me1/2 detected with no H3K4me3 (Fig. B). Dilution of the cultures, right triangle at the top of each column. Plates were incubated at 30 °C for 42 h prior to imaging. All cultures shown in the figure were grown on the same plate. The images were cut to remove a strain not being considered here

Article Snippet: The following antibodies were used; α-H3, Abcam ab1791 (4 µg/IP, lot #s: GR3297884-1, GR3297878-1, GR3356864-1 and GR3366670-1), α-H3K4me3, Epicypher 13-0041 (2 µg/IP, lot #s: 20083002-42 and 20218003-49), α-H3K4me2, Epicypher 13-0027 (4 µg/IP lot #: 20252002-04), α-H3K4me1, Epicypher 13-0040 (2 µg/IP lot #s: 19338001-42 and 20178005-44).

Techniques: Methylation, Activity Assay, Expressing, Mutagenesis, Western Blot, Incubation, Imaging

Journal: Current Genetics

Article Title: Histone 3 lysine 4 monomethylation supports activation of transcription in S. cerevisiae during nutrient stress

doi: 10.1007/s00294-022-01226-2

Figure Lengend Snippet: Distribution of H3K4me marks at three positions of the HIS3 gene. A Schematic showing the regions of HIS3 gene (promoter, 5′ ORF and 3′ ORF) evaluated by qPCR after ChIP for H3K4me1, H3K4me2 and H3K4me3. B Key to graph. C Association of H3K4me at the HIS3 promoter, 5′ ORF and 3′ ORF in yeast cultures grown in SC-His. D Association of H3K4me at the HIS3 promoter, 5′ ORF and 3′ ORF in yeast cultures grown in SC-HIS + 10 mM 3AT. See Supplementary Table 3 for statistical analysis; error bars, (± SEM, n = 4)

Article Snippet: The following antibodies were used; α-H3, Abcam ab1791 (4 µg/IP, lot #s: GR3297884-1, GR3297878-1, GR3356864-1 and GR3366670-1), α-H3K4me3, Epicypher 13-0041 (2 µg/IP, lot #s: 20083002-42 and 20218003-49), α-H3K4me2, Epicypher 13-0027 (4 µg/IP lot #: 20252002-04), α-H3K4me1, Epicypher 13-0040 (2 µg/IP lot #s: 19338001-42 and 20178005-44).

Techniques: