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: eLife

Article Title: Crystal structure of dopamine receptor D4 bound to the subtype selective ligand, L745870

doi: 10.7554/elife.48822

Figure Lengend Snippet: Figure 1. The overall structure of mouse DRD4 in complex with L745870. (a) Ribbon representation of mouse DRD4 (TM domain presented in cyan, ligand in yellow, and BRIL fusion in orange), as viewed parallel to the membrane with approximate membrane boundaries indicated with gray lines. (b) Top view of the mouse DRD4 structure. Rotation angle between Mol-A and Mol-B calculated with PyMOL is shown as a red angle sign. (c) The L745870 antagonist is shown in stick representation with carbon, nitrogen and chloride atoms shown in yellow, blue, and green, respectively. FoFc omit density map for L745870 is contoured at 2.5 s. (Also see Figure 1—figure supplement 5) (d) Chemical structure of L745870. The online version of this article includes the following figure supplement(s) for figure 1:

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Mouse) DRD4 SUNGENE BIOTECH GenBank: BC051421.1 Cell line (Spodoptera frugiperda) Sf9 and High5 Thermo Fisher Scientific Sf9: B825-01 High5:B85502 Cell line (Homo sapiens) HEK293t ATCC catalog numbers: CRL-3216 Transfected construct (Spodoptera frugiperda) pFastBac1-DRD4-BRIL This paper Crystal construct Transfected construct (Homo sapiens) pcDNA3.1-DRD4-cpGFP This paper Fluorescent sensor-based ligand-binding assay Continued on next page Zhou et al. eLife 2019;8:e48822.

Techniques: Membrane

Journal: eLife

Article Title: Crystal structure of dopamine receptor D4 bound to the subtype selective ligand, L745870

doi: 10.7554/elife.48822

Figure Lengend Snippet: Figure 2. Molecular details of the L745870-binding site in DRD4. (a) Mesh (Mol-A) and surface (Mol-B) representation of mouse DRD4 as viewed parallel to the membrane plane, clipped to reveal L745870 (colored as in Figure 1). (b) Interactions between the residues confining the binding pocket and L745870 (shown in magenta), with potential polar interactions depicted as dashed red lines. (c, d) Top view of surface representation of binding pocket in DRD2/3 and DRD4, respectively. L745870 is colored as in Figure 1. (e) Structural differences in the extended binding pockets (EBPs) of DRD4 (cyan), Figure 2 continued on next page

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Mouse) DRD4 SUNGENE BIOTECH GenBank: BC051421.1 Cell line (Spodoptera frugiperda) Sf9 and High5 Thermo Fisher Scientific Sf9: B825-01 High5:B85502 Cell line (Homo sapiens) HEK293t ATCC catalog numbers: CRL-3216 Transfected construct (Spodoptera frugiperda) pFastBac1-DRD4-BRIL This paper Crystal construct Transfected construct (Homo sapiens) pcDNA3.1-DRD4-cpGFP This paper Fluorescent sensor-based ligand-binding assay Continued on next page Zhou et al. eLife 2019;8:e48822.

Techniques: Binding Assay, Membrane

Journal: eLife

Article Title: Crystal structure of dopamine receptor D4 bound to the subtype selective ligand, L745870

doi: 10.7554/elife.48822

Figure Lengend Snippet: Figure 3. Interface details of mouse DRD4 in the asymmetric dimer. (a) Overview of the interface of the mouse DRD4 dimer. Dot representations of the interacting residues are shown for the transmembrane domain (gray) and exo-membrane regions (colored). (b, c) Extracellular and intracellular faces of the DRD4 dimer. Red dashed lines depict potential polar interactions. The online version of this article includes the following figure supplement(s) for figure 3:

Article Snippet: Key resources table Reagent type (species) or resource Designation Source or reference Identifiers Additional information Gene (Mouse) DRD4 SUNGENE BIOTECH GenBank: BC051421.1 Cell line (Spodoptera frugiperda) Sf9 and High5 Thermo Fisher Scientific Sf9: B825-01 High5:B85502 Cell line (Homo sapiens) HEK293t ATCC catalog numbers: CRL-3216 Transfected construct (Spodoptera frugiperda) pFastBac1-DRD4-BRIL This paper Crystal construct Transfected construct (Homo sapiens) pcDNA3.1-DRD4-cpGFP This paper Fluorescent sensor-based ligand-binding assay Continued on next page Zhou et al. eLife 2019;8:e48822.

Techniques: Membrane

Journal: eLife

Article Title: The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition

doi: 10.7554/elife.96850

Figure Lengend Snippet: Figure 1. DNA hypomethylation results in the activation of L1TD1 expression and loss of L1TD1 affects cell viability in HAP1 cells. (A) Quantification of DNA methylation levels at the L1TD1 promoter in HAP1 wildtype (WT), DNMT1 KO, and DNMT1/L1TD1 DKO cells using the MethyLight assay. DNA methylation is shown as percentage of methylation ratio (PMR). (B) qRT-PCR analysis of L1TD1 mRNA expression in HAP1 WT, DNMT1 KO, and DNMT1/ L1TD1 DKO cells. GAPDH was used as a normalization control and relative L1TD1 mRNA levels in DNMT1 KO cells were set to 1. Data are shown as a

Article Snippet: DOI: https://doi.org/10.7554/eLife.96850 13 of 22 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody DNMT1 (H- 300) (polyclonal rabbit) Santa Cruz sc20701 RRID:AB_2293064 1:1000 Antibody ɣ-H2AX (monoclonal mouse) Millipore JBW301 RRID:AB_2847865 1:1000 Antibody Histone H3 C- term (polyclonal rabbit) Abcam Ab1791 RRID:AB_302613 1:5000 Antibody Cleaved caspase 3 (polyclonal rabbit) Cell Signaling 9661 RRID:AB_2341188 1:50 Antibody Anti- mouse HRP (polyclonal) Jackson Laboratories 115-035-008 RRID:AB_2313585 1:10,000 Antibody Anti- rabbit HRP (polyclonal) Jackson Laboratories 211- 032- 171 RRID:AB_2339149 1:10,000 Antibody Goat anti- Rabbit IgG (H+L) Alexa Fluor Plus 488 Invitrogen A32731 RRID:AB_2633280 1:500 Antibody Goat anti- Mouse IgG (H+L) Alexa Fluor Plus 546 Invitrogen A11030 RRID:AB_2737024 1:500 Sequence- based reagent hL1TD1 _f This paper PCR primers CTTA CCCT GG TAGC CGAC CT Sequence- based reagent hL1TD1 _r This paper PCR primers GGCT GGCA AA TTTT CTAA GG Sequence- based reagent hARMC1_f This paper PCR primers AGCT CTGG AG CGAA TTTA AGA Sequence- based reagent hARMC1_r This paper PCR primers GGCA GACA TC CCTG ATCC TG Sequence- based reagent hYY2_f This paper PCR primers TCCC GGAT AG CATT GAAG AC Sequence- based reagent hYY2_r This paper PCR primers TTGA CCTG CA TTTG CTTC TG Sequence- based reagent hORF1p_f This paper PCR primers AGTG CTTA AAG GAGC TGAT GG Sequence- based reagent hORF1p_r This paper PCR primers AACT GGAA GAA AGGG TATC AGC Commercial assay or kit CellTiter- Glo Luminiscent Cell Viability Assay Promega G7571 Commercial assay or kit Monarch RNA Cleanup Kit New England Biolabs T2047L Commercial assay or kit Qubit RNA High Sensitivity kit Thermo Fisher Scientific Q32852 Commercial assay or kit iScript cDNA synthesis Kit Bio- Rad 1708891 Commercial assay or kit Wizard Genomic DNA isolation kit Promega Commercial assay or kit EZ DNA Methylation Kit Zymo Research D5001 Software, algorithm DESeq2 Love et al., 2014 RRID:SCR_015687 Software, algorithm TEtranscript Jin et al., 2015 RRID:SCR_023208 Continued on next page Continued Kavaklioglu et al. eLife 2024;13:RP96850.

Techniques: Activation Assay, Expressing, DNA Methylation Assay, Methylation, Quantitative RT-PCR, Control

Journal: eLife

Article Title: The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition

doi: 10.7554/elife.96850

Figure Lengend Snippet: Figure 3. L1TD1 cross-talk with its ancestor L1 ORF1p. (A) Volcano plot displaying the comparison of the proteomes of HAP1 DNMT1 KO and DNMT1/ L1TD1 DKO cells determined by mass spectrometry. Differentially abundant proteins were plotted as DNMT1/L1TD1 DKO over DNMT1 KO (log2FC ≥1, adj. p-value<0.05 [red] and log2FC ≤ –1, adj. p-value<0.05 [blue]). (B) Volcano plot illustrating the DESeq2 analysis of RNA-seq performed with HAP1 DNMT1 KO and DNMT1/L1TD1 DKO cells. Differentially expressed genes are plotted as DNMT1/L1TD1 DKO over DNMT1 KO (log2FC ≥1,

Article Snippet: DOI: https://doi.org/10.7554/eLife.96850 13 of 22 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody DNMT1 (H- 300) (polyclonal rabbit) Santa Cruz sc20701 RRID:AB_2293064 1:1000 Antibody ɣ-H2AX (monoclonal mouse) Millipore JBW301 RRID:AB_2847865 1:1000 Antibody Histone H3 C- term (polyclonal rabbit) Abcam Ab1791 RRID:AB_302613 1:5000 Antibody Cleaved caspase 3 (polyclonal rabbit) Cell Signaling 9661 RRID:AB_2341188 1:50 Antibody Anti- mouse HRP (polyclonal) Jackson Laboratories 115-035-008 RRID:AB_2313585 1:10,000 Antibody Anti- rabbit HRP (polyclonal) Jackson Laboratories 211- 032- 171 RRID:AB_2339149 1:10,000 Antibody Goat anti- Rabbit IgG (H+L) Alexa Fluor Plus 488 Invitrogen A32731 RRID:AB_2633280 1:500 Antibody Goat anti- Mouse IgG (H+L) Alexa Fluor Plus 546 Invitrogen A11030 RRID:AB_2737024 1:500 Sequence- based reagent hL1TD1 _f This paper PCR primers CTTA CCCT GG TAGC CGAC CT Sequence- based reagent hL1TD1 _r This paper PCR primers GGCT GGCA AA TTTT CTAA GG Sequence- based reagent hARMC1_f This paper PCR primers AGCT CTGG AG CGAA TTTA AGA Sequence- based reagent hARMC1_r This paper PCR primers GGCA GACA TC CCTG ATCC TG Sequence- based reagent hYY2_f This paper PCR primers TCCC GGAT AG CATT GAAG AC Sequence- based reagent hYY2_r This paper PCR primers TTGA CCTG CA TTTG CTTC TG Sequence- based reagent hORF1p_f This paper PCR primers AGTG CTTA AAG GAGC TGAT GG Sequence- based reagent hORF1p_r This paper PCR primers AACT GGAA GAA AGGG TATC AGC Commercial assay or kit CellTiter- Glo Luminiscent Cell Viability Assay Promega G7571 Commercial assay or kit Monarch RNA Cleanup Kit New England Biolabs T2047L Commercial assay or kit Qubit RNA High Sensitivity kit Thermo Fisher Scientific Q32852 Commercial assay or kit iScript cDNA synthesis Kit Bio- Rad 1708891 Commercial assay or kit Wizard Genomic DNA isolation kit Promega Commercial assay or kit EZ DNA Methylation Kit Zymo Research D5001 Software, algorithm DESeq2 Love et al., 2014 RRID:SCR_015687 Software, algorithm TEtranscript Jin et al., 2015 RRID:SCR_023208 Continued on next page Continued Kavaklioglu et al. eLife 2024;13:RP96850.

Techniques: Comparison, Mass Spectrometry, RNA Sequencing

Journal: eLife

Article Title: The domesticated transposon protein L1TD1 associates with its ancestor L1 ORF1p to promote LINE-1 retrotransposition

doi: 10.7554/elife.96850

Figure Lengend Snippet: Figure 4. L1TD1 promotes L1 retrotransposition. (A) Schematic representation of plasmids used for retrotransposition (figure modified from Kopera et al., 2016 and generated with BioRender.com). The pJJ101/L1.3 construct contains the full-length human L1.3 element with a blasticidin deaminase gene (mblast) inserted in antisense within the 3’UTR. The mblast gene is disrupted by an intron and mblast expression occurs only when L1 transcript is expressed, reverse transcribed, and inserted into the genome. The pJJ105/L1.3 plasmid contains a mutation in the reverse transcriptase (RT), resulting in defective retrotransposition. The backbone plasmid pCEP4 was used as additional negative control. The blasticidin deaminase gene containing plasmid pLenti6.2 was used as transfection/selection control. (B) Workflow of retrotransposition assay. DNMT1 KO and DNMT1/L1TD1 DKO cells were separately transfected with pJJ101 and control plasmids. Equal number of cells were seeded for each condition. Blasticidin selection (10 µg/ml) was started at day 4 and resistant colonies were counted on day 13. This panel was created using BioRender.com. (C) Bar graph showing the average number of retrotransposition events per 106 cells seeded in three independent experiments. Blasticidin-resistant colonies in pLenti6.2 transfected cells were used for normalization. Statistical significance was determined using unpaired t-test. All data in the figure are shown as a mean of ± SD of three independent experiments, ****p≤0.0001. (D) Representative pictures of bromophenol blue stainings of blasticidin-resistant colonies for each genotype and each transfection.

Article Snippet: DOI: https://doi.org/10.7554/eLife.96850 13 of 22 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Antibody DNMT1 (H- 300) (polyclonal rabbit) Santa Cruz sc20701 RRID:AB_2293064 1:1000 Antibody ɣ-H2AX (monoclonal mouse) Millipore JBW301 RRID:AB_2847865 1:1000 Antibody Histone H3 C- term (polyclonal rabbit) Abcam Ab1791 RRID:AB_302613 1:5000 Antibody Cleaved caspase 3 (polyclonal rabbit) Cell Signaling 9661 RRID:AB_2341188 1:50 Antibody Anti- mouse HRP (polyclonal) Jackson Laboratories 115-035-008 RRID:AB_2313585 1:10,000 Antibody Anti- rabbit HRP (polyclonal) Jackson Laboratories 211- 032- 171 RRID:AB_2339149 1:10,000 Antibody Goat anti- Rabbit IgG (H+L) Alexa Fluor Plus 488 Invitrogen A32731 RRID:AB_2633280 1:500 Antibody Goat anti- Mouse IgG (H+L) Alexa Fluor Plus 546 Invitrogen A11030 RRID:AB_2737024 1:500 Sequence- based reagent hL1TD1 _f This paper PCR primers CTTA CCCT GG TAGC CGAC CT Sequence- based reagent hL1TD1 _r This paper PCR primers GGCT GGCA AA TTTT CTAA GG Sequence- based reagent hARMC1_f This paper PCR primers AGCT CTGG AG CGAA TTTA AGA Sequence- based reagent hARMC1_r This paper PCR primers GGCA GACA TC CCTG ATCC TG Sequence- based reagent hYY2_f This paper PCR primers TCCC GGAT AG CATT GAAG AC Sequence- based reagent hYY2_r This paper PCR primers TTGA CCTG CA TTTG CTTC TG Sequence- based reagent hORF1p_f This paper PCR primers AGTG CTTA AAG GAGC TGAT GG Sequence- based reagent hORF1p_r This paper PCR primers AACT GGAA GAA AGGG TATC AGC Commercial assay or kit CellTiter- Glo Luminiscent Cell Viability Assay Promega G7571 Commercial assay or kit Monarch RNA Cleanup Kit New England Biolabs T2047L Commercial assay or kit Qubit RNA High Sensitivity kit Thermo Fisher Scientific Q32852 Commercial assay or kit iScript cDNA synthesis Kit Bio- Rad 1708891 Commercial assay or kit Wizard Genomic DNA isolation kit Promega Commercial assay or kit EZ DNA Methylation Kit Zymo Research D5001 Software, algorithm DESeq2 Love et al., 2014 RRID:SCR_015687 Software, algorithm TEtranscript Jin et al., 2015 RRID:SCR_023208 Continued on next page Continued Kavaklioglu et al. eLife 2024;13:RP96850.

Techniques: Modification, Generated, Construct, Expressing, Reverse Transcription, Plasmid Preparation, Mutagenesis, Negative Control, Transfection, Selection, Control

Journal: Journal of Virology

Article Title: CD63 Regulates Epstein-Barr Virus LMP1 Exosomal Packaging, Enhancement of Vesicle Production, and Noncanonical NF-κB Signaling

doi: 10.1128/JVI.02251-16

Figure Lengend Snippet: LMP1 trafficking to lipid rafts and downstream signaling do not require CD63. (A) HEK293 control and CD63 knockout cells were transfected with GFP-LMP1. Lipid rafts were biochemically isolated for immunoblot analysis of whole-cell (input) and lipid raft-associated proteins, equal masses loaded. (B) Cytoplasmic and nuclear fractions of HEK293 control and CD63 CRISPR cells transfected with GFP or GFP-LMP1 were separated and confirmed by enrichment of cytoplasmic calnexin or nuclear histone H4 protein, equal masses loaded. (C) Akt and ERK activation in cytoplasmic fractions of HEK293 and CD63 CRISPR cells following GFP-LMP1 transfection was measured, equal masses loaded. Relative levels of phospho-Akt and phospho-ERK were averaged over three independent experiments. (D) HA-LMP1 packaging in EVs (equal volumes) from Rat1 cells stably expressing a pBabe-HA-LMP1 vector following CD63 knockout. (E) Quantitation of HA-LMP1 packaging in Rat1 EVs from three independent experiments. (F) Focus formation assay was performed using Rat1 control or CD63 knockout cells transduced with an empty pBabe vector or pBabe-HA-LMP1. (G and H) Immunoblot analysis of LMP1-induced NF-κB signaling activation in cytoplasmic (G) and nuclear (H) fractions of HEK293 control and CD63 CRISPR cells, equal masses loaded. The results shown are representative of findings from multiple experiments.

Article Snippet: Blots were probed with primary antibodies against the following: Alix (Q-19; Santa Cruz Biotechnology), HSC70 (B-6; Santa Cruz), TSG101 (C-2; Santa Cruz), calnexin (11397; Santa Cruz), caveolin-1 (D46G3; Cell Signaling), flotillin-2 (H-90; Santa Cruz), CD63 (TS63; Abcam), GFP (600-101-215; Rockland), HA (C29F4; Cell Signaling), LMP1 (CS1-4; Dako), histone H4 ( 81 , 82 ), IKKα (11930; Cell Signaling), IKKβ (8943; Cell Signaling), phospho-IKKα/β (2697; Cell Signaling), IκBα (4814; Cell Signaling), phospho-IκBα (2859; Cell Signaling), NF-κB p65 (8242; Cell Signaling), phospho-NF-κB p65 (3033; Cell Signaling), RelB (4922; Cell Signaling), NF-κB p100/p52 (3017; Cell Signaling), phospho-NF-κB p100/p52 (4810; Cell Signaling), ERK 2 (C-14; Santa Cruz), phospho-p44/42 MAPK (9106; Cell Signaling), Akt (9272; Cell Signaling), phospho-Akt (4060; Cell Signaling), and phospho-STAT3 (9134; Cell Signaling).

Techniques: Knock-Out, Transfection, Isolation, Western Blot, CRISPR, Activation Assay, Stable Transfection, Expressing, Plasmid Preparation, Quantitation Assay, Tube Formation Assay, Transduction