Journal: Neuron

Article Title: Age-dependent effects of apoE reduction using antisense oligonucleotides in a model of β-amyloidosis

doi: 10.1016/j.neuron.2017.11.014

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level was used as a reference (Mm99999915_g1 Gapdh).

Techniques: Purification, Virus, Recombinant, Bicinchoninic Acid Protein Assay, Sequencing, Control, Software

Journal:

Article Title: Characterization of an Endoprotease (PrpL) Encoded by a PvdS-Regulated Gene in Pseudomonas aeruginosa

doi: 10.1128/IAI.69.9.5385-5394.2001

Figure Lengend Snippet: Strains, plasmids, and primers used in this study

Article Snippet: Antibiotics were used at the following concentrations: for Escherichia coli , ampicillin at 100 μg/ml, gentamicin at 15 μg/ml, kanamycin at 100 μg/ml, and tetracycline at 15 μg/ml; for P. aeruginosa , carbenicillin at 500 μg/ml, gentamicin at 75 μg/ml, and tetracycline at 150 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain, plasmid, or primer Genotype or characteristic(s) a Source or reference Strains P. aeruginosa PAO1 (ATCC 15692) Prototroph, wound isolate 23 PAO1 Δ prpL :: Gm PAO1 with a Gm r cassette replacing a region of prpL This study PAO1 Δ pvdS :: Gm PAO1 with a Gm r cassette replacing a region of pvdS 33 PAO1 Δ ptxR :: Gm PAO1 with a Gm r cassette replacing a region of ptxR 50 PA103 Prototroph 28 PAK Prototroph 32 PA14 Human clinical isolate 40 Ps388 Prototroph, hypotoxigenic 53 WR5 Prototroph, exotoxin A deficient 53 M2 Mouse strain 49 CF1-1, CF2-1, CF3-1 CF isolates from Denver, Colo.

Techniques: Plasmid Preparation, Clone Assay, TA Cloning, Conjugation Assay, Expressing

Journal:

Article Title: Characterization of an Endoprotease (PrpL) Encoded by a PvdS-Regulated Gene in Pseudomonas aeruginosa

doi: 10.1128/IAI.69.9.5385-5394.2001

Figure Lengend Snippet: Effects of PvdS on pyoverdine production and proteolytic and elastolytic activities a

Article Snippet: Antibiotics were used at the following concentrations: for Escherichia coli , ampicillin at 100 μg/ml, gentamicin at 15 μg/ml, kanamycin at 100 μg/ml, and tetracycline at 15 μg/ml; for P. aeruginosa , carbenicillin at 500 μg/ml, gentamicin at 75 μg/ml, and tetracycline at 150 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain, plasmid, or primer Genotype or characteristic(s) a Source or reference Strains P. aeruginosa PAO1 (ATCC 15692) Prototroph, wound isolate 23 PAO1 Δ prpL :: Gm PAO1 with a Gm r cassette replacing a region of prpL This study PAO1 Δ pvdS :: Gm PAO1 with a Gm r cassette replacing a region of pvdS 33 PAO1 Δ ptxR :: Gm PAO1 with a Gm r cassette replacing a region of ptxR 50 PA103 Prototroph 28 PAK Prototroph 32 PA14 Human clinical isolate 40 Ps388 Prototroph, hypotoxigenic 53 WR5 Prototroph, exotoxin A deficient 53 M2 Mouse strain 49 CF1-1, CF2-1, CF3-1 CF isolates from Denver, Colo.

Techniques: Activity Assay

Journal:

Article Title: Characterization of an Endoprotease (PrpL) Encoded by a PvdS-Regulated Gene in Pseudomonas aeruginosa

doi: 10.1128/IAI.69.9.5385-5394.2001

Figure Lengend Snippet: Profiles of extracellular proteins from P. aeruginosa PAO1 and ΔpvdS::Gm. The arrow indicates the 27-kDa protein that was investigated in this study. The molecular masses of the proteins in the marker lane (M) are indicated to the left.

Article Snippet: Antibiotics were used at the following concentrations: for Escherichia coli , ampicillin at 100 μg/ml, gentamicin at 15 μg/ml, kanamycin at 100 μg/ml, and tetracycline at 15 μg/ml; for P. aeruginosa , carbenicillin at 500 μg/ml, gentamicin at 75 μg/ml, and tetracycline at 150 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain, plasmid, or primer Genotype or characteristic(s) a Source or reference Strains P. aeruginosa PAO1 (ATCC 15692) Prototroph, wound isolate 23 PAO1 Δ prpL :: Gm PAO1 with a Gm r cassette replacing a region of prpL This study PAO1 Δ pvdS :: Gm PAO1 with a Gm r cassette replacing a region of pvdS 33 PAO1 Δ ptxR :: Gm PAO1 with a Gm r cassette replacing a region of ptxR 50 PA103 Prototroph 28 PAK Prototroph 32 PA14 Human clinical isolate 40 Ps388 Prototroph, hypotoxigenic 53 WR5 Prototroph, exotoxin A deficient 53 M2 Mouse strain 49 CF1-1, CF2-1, CF3-1 CF isolates from Denver, Colo.

Techniques: Marker

Journal:

Article Title: Characterization of an Endoprotease (PrpL) Encoded by a PvdS-Regulated Gene in Pseudomonas aeruginosa

doi: 10.1128/IAI.69.9.5385-5394.2001

Figure Lengend Snippet: Alignment of the P. aeruginosa (PA) and L. enzymogenes (LE) endoproteases. Regions of identity are boxed. The single-stemmed arrow indicates the predicted signal cleavage site, and the double-stemmed arrow indicates the proenzyme junction in L. enzymogenes. The diamonds indicate the catalytic triad in the L. enzymogenes endoprotease, and the asterisk indicates a stop codon. The RGD motif in the P. aeruginosa endoprotease is indicated by the black bar.

Article Snippet: Antibiotics were used at the following concentrations: for Escherichia coli , ampicillin at 100 μg/ml, gentamicin at 15 μg/ml, kanamycin at 100 μg/ml, and tetracycline at 15 μg/ml; for P. aeruginosa , carbenicillin at 500 μg/ml, gentamicin at 75 μg/ml, and tetracycline at 150 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain, plasmid, or primer Genotype or characteristic(s) a Source or reference Strains P. aeruginosa PAO1 (ATCC 15692) Prototroph, wound isolate 23 PAO1 Δ prpL :: Gm PAO1 with a Gm r cassette replacing a region of prpL This study PAO1 Δ pvdS :: Gm PAO1 with a Gm r cassette replacing a region of pvdS 33 PAO1 Δ ptxR :: Gm PAO1 with a Gm r cassette replacing a region of ptxR 50 PA103 Prototroph 28 PAK Prototroph 32 PA14 Human clinical isolate 40 Ps388 Prototroph, hypotoxigenic 53 WR5 Prototroph, exotoxin A deficient 53 M2 Mouse strain 49 CF1-1, CF2-1, CF3-1 CF isolates from Denver, Colo.

Techniques:

Journal:

Article Title: Characterization of an Endoprotease (PrpL) Encoded by a PvdS-Regulated Gene in Pseudomonas aeruginosa

doi: 10.1128/IAI.69.9.5385-5394.2001

Figure Lengend Snippet: Protease activities of P. aeruginosa PAO1, ΔpvdS::Gm, and ΔprpL::Gm. Strains were grown in D-TSB containing 1% glycerol and 50 mM glutamate. Addition of FeCl3 to a final concentration of 50 μg/ml was used for the high-iron medium. Cultures were incubated with shaking at 32°C for 15 h. Extracellular proteins were precipitated with ammonium sulfate. Protease activity was determined by spotting the precipitated proteins onto D-BHI skim milk agar plates (48) and measuring the zone of hydrolysis produced after incubating the plates at 37°C for 24 to 38 h. The disk diameter is 6.5 mm.

Article Snippet: Antibiotics were used at the following concentrations: for Escherichia coli , ampicillin at 100 μg/ml, gentamicin at 15 μg/ml, kanamycin at 100 μg/ml, and tetracycline at 15 μg/ml; for P. aeruginosa , carbenicillin at 500 μg/ml, gentamicin at 75 μg/ml, and tetracycline at 150 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain, plasmid, or primer Genotype or characteristic(s) a Source or reference Strains P. aeruginosa PAO1 (ATCC 15692) Prototroph, wound isolate 23 PAO1 Δ prpL :: Gm PAO1 with a Gm r cassette replacing a region of prpL This study PAO1 Δ pvdS :: Gm PAO1 with a Gm r cassette replacing a region of pvdS 33 PAO1 Δ ptxR :: Gm PAO1 with a Gm r cassette replacing a region of ptxR 50 PA103 Prototroph 28 PAK Prototroph 32 PA14 Human clinical isolate 40 Ps388 Prototroph, hypotoxigenic 53 WR5 Prototroph, exotoxin A deficient 53 M2 Mouse strain 49 CF1-1, CF2-1, CF3-1 CF isolates from Denver, Colo.

Techniques: Concentration Assay, Incubation, Activity Assay, Produced

Journal:

Article Title: Characterization of an Endoprotease (PrpL) Encoded by a PvdS-Regulated Gene in Pseudomonas aeruginosa

doi: 10.1128/IAI.69.9.5385-5394.2001

Figure Lengend Snippet: Demonstration of PrpL activity. (A) Approximately 10 μg of lactoferrin (LF) was digested with proteins from supernatants of P. aeruginosa ΔprpL::Gm or PAO1 at 37°C for 15, 30, and 60 min. Digestion products were analyzed by SDS-PAGE. (B) Approximately 10 μg of lactoferrin (LF) was digested with proteins from supernatants of P. aeruginosa ΔprpL::Gm or PAO1 for 60 min at 23, 32, 37, or 42°C. Digestion products were separated by SDS-PAGE. (C) Approximately 10 μg of lactoferrin (LF) was digested with proteins from supernatants of P. aeruginosa ΔprpL::Gm or PAO1 at 37°C for 60 min. Products were separated by SDS-PAGE, transferred to nitrocellulose, and probed for LF. (D) Approximately 10 μg of transferrin (TF) was digested with proteins from supernatants of P. aeruginosa ΔprpL::Gm or PAO1 at 37°C for 15, 30, and 60 min. Digestion products were analyzed by SDS-PAGE. (E) Approximately 10 μg of decorin (D) was digested with proteins from supernatants of P. aeruginosa ΔprpL::Gm or PAO1 at 37°C for 60 min. Digestion products were analyzed by SDS-PAGE. Lanes M contained molecular size markers.

Article Snippet: Antibiotics were used at the following concentrations: for Escherichia coli , ampicillin at 100 μg/ml, gentamicin at 15 μg/ml, kanamycin at 100 μg/ml, and tetracycline at 15 μg/ml; for P. aeruginosa , carbenicillin at 500 μg/ml, gentamicin at 75 μg/ml, and tetracycline at 150 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain, plasmid, or primer Genotype or characteristic(s) a Source or reference Strains P. aeruginosa PAO1 (ATCC 15692) Prototroph, wound isolate 23 PAO1 Δ prpL :: Gm PAO1 with a Gm r cassette replacing a region of prpL This study PAO1 Δ pvdS :: Gm PAO1 with a Gm r cassette replacing a region of pvdS 33 PAO1 Δ ptxR :: Gm PAO1 with a Gm r cassette replacing a region of ptxR 50 PA103 Prototroph 28 PAK Prototroph 32 PA14 Human clinical isolate 40 Ps388 Prototroph, hypotoxigenic 53 WR5 Prototroph, exotoxin A deficient 53 M2 Mouse strain 49 CF1-1, CF2-1, CF3-1 CF isolates from Denver, Colo.

Techniques: Activity Assay, SDS Page

Journal:

Article Title: Characterization of an Endoprotease (PrpL) Encoded by a PvdS-Regulated Gene in Pseudomonas aeruginosa

doi: 10.1128/IAI.69.9.5385-5394.2001

Figure Lengend Snippet: Proteins from culture supernatants from various P. aeruginosa strains were precipitated and analyzed by SDS-PAGE and for protease activity on BHI skim milk plates. The arrowhead indicates the PrpL band. The molecular masses of the markers (lane M) are indicated to the left.

Article Snippet: Antibiotics were used at the following concentrations: for Escherichia coli , ampicillin at 100 μg/ml, gentamicin at 15 μg/ml, kanamycin at 100 μg/ml, and tetracycline at 15 μg/ml; for P. aeruginosa , carbenicillin at 500 μg/ml, gentamicin at 75 μg/ml, and tetracycline at 150 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain, plasmid, or primer Genotype or characteristic(s) a Source or reference Strains P. aeruginosa PAO1 (ATCC 15692) Prototroph, wound isolate 23 PAO1 Δ prpL :: Gm PAO1 with a Gm r cassette replacing a region of prpL This study PAO1 Δ pvdS :: Gm PAO1 with a Gm r cassette replacing a region of pvdS 33 PAO1 Δ ptxR :: Gm PAO1 with a Gm r cassette replacing a region of ptxR 50 PA103 Prototroph 28 PAK Prototroph 32 PA14 Human clinical isolate 40 Ps388 Prototroph, hypotoxigenic 53 WR5 Prototroph, exotoxin A deficient 53 M2 Mouse strain 49 CF1-1, CF2-1, CF3-1 CF isolates from Denver, Colo.

Techniques: SDS Page, Activity Assay

Journal:

Article Title: Characterization of an Endoprotease (PrpL) Encoded by a PvdS-Regulated Gene in Pseudomonas aeruginosa

doi: 10.1128/IAI.69.9.5385-5394.2001

Figure Lengend Snippet: Persistence of P. aeruginosa PAO1 and Δ prpL :: Gm in an agarose bead rat lung model

Article Snippet: Antibiotics were used at the following concentrations: for Escherichia coli , ampicillin at 100 μg/ml, gentamicin at 15 μg/ml, kanamycin at 100 μg/ml, and tetracycline at 15 μg/ml; for P. aeruginosa , carbenicillin at 500 μg/ml, gentamicin at 75 μg/ml, and tetracycline at 150 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain, plasmid, or primer Genotype or characteristic(s) a Source or reference Strains P. aeruginosa PAO1 (ATCC 15692) Prototroph, wound isolate 23 PAO1 Δ prpL :: Gm PAO1 with a Gm r cassette replacing a region of prpL This study PAO1 Δ pvdS :: Gm PAO1 with a Gm r cassette replacing a region of pvdS 33 PAO1 Δ ptxR :: Gm PAO1 with a Gm r cassette replacing a region of ptxR 50 PA103 Prototroph 28 PAK Prototroph 32 PA14 Human clinical isolate 40 Ps388 Prototroph, hypotoxigenic 53 WR5 Prototroph, exotoxin A deficient 53 M2 Mouse strain 49 CF1-1, CF2-1, CF3-1 CF isolates from Denver, Colo.

Techniques:

Journal:

Article Title: Characterization of an Endoprotease (PrpL) Encoded by a PvdS-Regulated Gene in Pseudomonas aeruginosa

doi: 10.1128/IAI.69.9.5385-5394.2001

Figure Lengend Snippet: Transcriptional analysis of prpL. (A) Promoter region of the prpL gene. The Shine-Dalgarno (S/D) site, the start site of the coding sequence, and the putative transcriptional start sites of T1 and T2 are indicated. The 422- and 727-nt probes used for RNase protection analysis of the prpL gene are shown by black bars. The consensus sequence for the proposed iron starvation box is indicated. (B) RNase protection analysis of P. aeruginosa PAO1 and ΔpvdS::Gm RNAs isolated at the time points shown from cells grown aerobically and microaerobically (5% oxygen) under low (−)- or high (+)-iron conditions and probed with a 422-base riboprobe. The positions of 100- and 200-base RNA size standards are indicated. (C) RNase protection analysis of P. aeruginosa PAO1, ΔpvdS::Gm, and ΔptxR::Gm. RNA was isolated at 10 h from cells grown aerobically under low (−)- and high (+)-iron conditions and probed with a 727-base riboprobe. The relative intensities of the transcripts in panels B and C were quantified with a Bio-Rad Personal FX phosphorimager using Quantity One software (version 4.0.3) from Bio-Rad. The sizes of the RNA fragments in the marker (M) lane are shown.

Article Snippet: Antibiotics were used at the following concentrations: for Escherichia coli , ampicillin at 100 μg/ml, gentamicin at 15 μg/ml, kanamycin at 100 μg/ml, and tetracycline at 15 μg/ml; for P. aeruginosa , carbenicillin at 500 μg/ml, gentamicin at 75 μg/ml, and tetracycline at 150 μg/ml. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain, plasmid, or primer Genotype or characteristic(s) a Source or reference Strains P. aeruginosa PAO1 (ATCC 15692) Prototroph, wound isolate 23 PAO1 Δ prpL :: Gm PAO1 with a Gm r cassette replacing a region of prpL This study PAO1 Δ pvdS :: Gm PAO1 with a Gm r cassette replacing a region of pvdS 33 PAO1 Δ ptxR :: Gm PAO1 with a Gm r cassette replacing a region of ptxR 50 PA103 Prototroph 28 PAK Prototroph 32 PA14 Human clinical isolate 40 Ps388 Prototroph, hypotoxigenic 53 WR5 Prototroph, exotoxin A deficient 53 M2 Mouse strain 49 CF1-1, CF2-1, CF3-1 CF isolates from Denver, Colo.

Techniques: Sequencing, Isolation, Software, Marker

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 1. GIV (CCDC88A) is highly expressed in spermatocytes in testis and localizes to the acrosomal cap. (A) Bar graph displays the relative fluorescence unit (RFU) of endogenous full-length GIV protein in immunoblots of organ lysates published previously using three independent anti-GIV antibodies raised against different epitopes of GIV (Anai et al., 2005). (Figure 1—source data 1)(B) RNA expression in the single-cell-type clusters identified in the human testis visualized by a UMAP plot (inset) and a bar plot. The bar plot shows RNA expression (pTPM) in each cell-type cluster.

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Fluorescence, Western Blot, RNA Expression

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 2. Transcripts of CCDC88A (GIV) are downregulated in infertile male testis and semen. (A) Schematic displays the approach used to search NCBI GEO database for testis and sperm transcriptomic datasets suitable to study correlations between the abundance of CCDC88A transcripts and male fertility. (B–E) Whisker plots show the relative abundance of CCDC88A (expressed as Log2 normalized expression; see Materials and methods for different normalization approaches used for microarray and RNA-seq datasets) in sperm or testis samples (as annotated using schematics) in samples

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Whisker Assay, Expressing, Microarray, RNA Sequencing

Journal: eLife

Article Title: GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

doi: 10.7554/elife.69160

Figure Lengend Snippet: Figure 8. Summary and working model: spatiotemporally segregated roles of GIV/Girdin during sperm capacitation. Schematic summarizes the key findings in this work and places them in the context of existing literature. GIV is likely to primarily function during capacitation of sperm, during which it fulfills two key roles as a signal transducer in a spatiotemporally segregated manner. The first role (right, top) is in the head of the sperm, where GIV’s GEM motif inhibits the AC→cAMP pathway and prevents acrosomal reaction. The second role (right, bottom) is in the mid-piece and tail region of the sperm, which involves tyrosine phosphorylation of GIV, which

Article Snippet: Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody Rabbit polyclonal anti- GIV (Girdin) (T- 13) Santa Cruz Biotechnology sc- 133371 Antibody Rabbit monoclonal diagnostic grade anti- Girdin/GIV antibody Custom; Sprint Bioscience SP173 Validated in prior publication Ghosh et al., 2016 Antibody Rabbit polyclonal anti- GIV (Girdin) (CC- Ab) Millipore Sigma ABT80 Antibody Rabbit polyclonal anti- GIV pS1675 Ab Custom, from 21t Century Biosciences n/a Validated in prior publication Bhandari et al., 2015 Antibody Rabbit polyclonal anti- GIV pS1689 Ab Custom, from 21st Century Biosciences n/a Validated in prior publication LópezSánchez et al., 2013 Antibody Rabbit monoclonal anti- GIV pY1764 Ab Custom, Spring Biosciences Inc n/a Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 Antibody Rabbit monoclonal antipT308 AKT Cell Signaling Technology D9E Antibody Mouse monoclonal anti- total AKT Cell Signaling Technology 40D4 Antibody Mouse anti- sp56 Thermo Fisher Scientific (Waltham, MA) MA1- 10866 Antibody Mouse anti- human hexokinase 1/2 monoclonal antibody R&D Systems, (Minneapolis, MN) MAB8179 Antibody Rabbit anti- phospho- PKA substrate (RRXS*/T*)100G7E Cell Signaling Technology 9624 Antibody Goat anti- rabbit IgG, Alexa Fluor 594 conjugated ThermoFisher Scientific A11072 For immunofluorescence (IF) Antibody Goat anti- mouse IgG, Alexa Fluor 488 conjugated ThermoFisher Scientific A11017 For immunofluorescence (IF) Antibody IRDye 800CW goat antimouse IgG secondary (1:10,000) LI- COR Biosciences 926- 32210 For immunoblotting Antibody IRDye 680RD goat anti- rabbit IgG secondary (1:10,000) LI- COR Biosciences 926- 68071 For immunoblotting Reynoso, Castillo, Katkar et al. eLife 2021;10:e69160.

Techniques: Phospho-proteomics

Journal: Science advances

Article Title: Human DNA topoisomerase I poisoning causes R loop-mediated genome instability attenuated by transcription factor IIS.

doi: 10.1126/sciadv.adm8196

Figure Lengend Snippet: Fig. 1. Top1ccs induce dynamic changes of hybrid levels at specific genomic sites in HCT116 cells. (A) Volcano plots of differential analyses of DRIP-seq signals be- tween cells treated with CPT for 5 min (5′ CPT) versus untreated cells (NT) (top) and cells treated with CPT for 60 min (60’ CPT) versus NT (bottom). Each dot represents a DRIP-seq peak. Axis values are log2(fold changes) of DRIP-seq levels (x axis) and −log10(P values) of limma test (y axis). Red dots indicate peaks with P value less than 0.01, shown as a dashed line. (B) Metaplots (left) and heatmap (right) of DRIP-seq normalized read density (n.r.d.) in seven R loop categories distinguished by different kinetics. Line colors of metaplots correspond to the means of two biological replicates as reported in the legend. Heatmap colors represent DRIP levels as in legend. In both meta- plots and heatmaps, “c” indicates DRIP peak center in a window of + or –3 kilo–base pairs (kbp). RH indicates DRIP samples treated with RNase H1 before immunopre- cipitation. (C) Fractions of DRIP peaks over genomic features, as shown in the legend. (D) Metaplots of DRIP-seq (5- and 60-min treatment with CPT), GRO-seq (38), RNAPII ChIP-seq (10-min treatment with CPT) (6), Top1 ChIP-seq (6), and Top1cc-seq (4-min treatment with CPT) (6) normalized levels for each R loop kinetic category. RSS is the start site of R loop peaks. The graph is in a window of −1/+8 kbp with respect to RSS. Line colors as in legend.

Article Snippet: MATERIALS AND METHODS Cell lines and treatment Human cancer HCT116 cells, U2OS, and HeLa cell lines were purchased from American Type Culture Collection (LGC Standards S.r.l., Milan, Italy).

Techniques: ChIP-sequencing

Journal: Science advances

Article Title: Human DNA topoisomerase I poisoning causes R loop-mediated genome instability attenuated by transcription factor IIS.

doi: 10.1126/sciadv.adm8196

Figure Lengend Snippet: Fig. 4. Top1ccs induce DSB clusters after short times in HCT116 cells. (A) Metaplot (left) and heat map (right) of END-seq levels normalized read density at transient, late, and persistent DSB regions, showing kinetics of DSBs induced by CPT-mediated Top1ccs at control conditions (NT) and 10 min (10′ CPT) and 20 min (20′ CPT) of treatment. Line colors as in legend. “c” indicates the center of the region in a window of ±3 kbp. All signals are reported as means of two biological replicates normalized levels. (B) Scatter plot of correlation between number of genes (y axis) and number of persistent DSBs (x axis) per chromosome. Spearman correlation coefficient and P values are reported. (C) Proportion of transient, late, and persistent DSBs over genomic features, as percentage. Colors as in legend. (D) Metaplot showing ChIP-seq normalized signals of euchromatin histone markers (H3K4me3, H3K4me2, H3K4me1, and H3K27ac), heterochromatin histone marker H3K9me2, and DNase-seq signals (39) at transient, late, and persistent DSB clusters, excluding those localized at intergenic loci. Histone marker ChIP-seq signal is reported as −log10(P value). DNase-seq signals are reported as normalized read-depth levels. “c” indicates the center of the region in a window of ±10 kbp. (E) Metaplot showing GRO-seq (nontreated), RNAPII ChIP-seq (nontreated and 10-min CPT-treated), and Top1cc-seq (4-min CPT) normalized levels at tran- sient, late, and persistent DSB regions, excluding regions localized at intergenic loci. Line colors as in legend. “c” indicates the center of the region in a window of ±5 kbp.

Article Snippet: MATERIALS AND METHODS Cell lines and treatment Human cancer HCT116 cells, U2OS, and HeLa cell lines were purchased from American Type Culture Collection (LGC Standards S.r.l., Milan, Italy).

Techniques: Control, ChIP-sequencing, Marker