Journal: bioRxiv

Article Title: A systematic evaluation of the design, orientation, and sequence context dependencies of massively parallel reporter assays

doi: 10.1101/576405

Figure Lengend Snippet: (A) Beeswarm plot of the Pearson correlation values for each of the three possible pairwise comparisons among the replicates of each MPRA technique. B) Scatter matrix displaying scatter plots corresponding to each of the 36 pairs of possible inter-assay comparisons (lower diagonal elements). Shown on the diagonal is a histogram of the log 2 (RNA/DNA) ratios, averaged among replicate samples. Also shown are Pearson correlation values among each pair of comparisons, with the size of the text proportional to the magnitude of the correlation coefficient (upper diagonal elements). See also for the equivalent result but with Spearman correlations. C) Principal components analysis (PCA) of 27 experiments, i.e. three replicates (labeled ‘1’, ‘2’, and ‘3’) of each of the nine different MPRA approaches tested. Shown are the first two principal components that together explain over half of the variation. Slight jitter was added to each data point to enhance readability. D) Violin plots displaying the distribution of average log 2 (RNA/DNA) ratios across independent transfections for positive controls, negative controls, and putative enhancer sequences tested, for each of the nine assays.

Article Snippet: To generate wild-type lentiviral libraries (5’/5’ WT, 5’/3’ WT, and 3’/3’ WT), the cells were co-transfected with 5.5 μg of lentiMPRA libraries, 1.85 μg of pMD2.G (Addgene 12259) and 3.65 μg of psPAX2 (Addgene 12260), which encodes a wild type pol , using EndoFectin Lenti transfection reagent (GeneCopoeia) according to manufacturer’s instruction.

Techniques: Inter Assay, Labeling, Transfection

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: Anti-HIV Activities and Mechanism of 12- O -Tricosanoylphorbol-20-acetate, a Novel Phorbol Ester from Ostodes katharinae

doi: 10.3390/molecules22091498

Figure Lengend Snippet: Hop-8 inhibited HIV-1 and HIV-2 wild-type, clinical isolates and resistant strains with low cytotoxicity. ( A ) The cytotoxicity of hop-8 in C8166; ( B ) The cytotoxicity of hop-8 in peripheral blood mononuclear cells (PBMCs); ( C ) The antiviral activities of hop-8 against the clinical isolates HIV-1 KM018 , and HIV-1 TC-1 were measured in PBMCs; ( D ) The antiviral activities of hop-8 against wild-type HIV-1 IIIB , HIV-2 CBL-20 , and HIV-1 ΔVif NL4-3 were measured in the C8166 cell line; ( E ) Antiviral activities of hop-8 against the HIV-1 drug resistant strains HIV-1 A17 , HIV-1 NL4-3 gp41 (36G) V38A, N42T , HIV-1 RF/V28F/184V , and HIV-1 74V . The levels of p24 in the cell culture supernatant were measured by ELISA. Each data point represents the mean percent inhibition (relative to the positive control) ± standard deviation (bars), n ≥ 2. Data were analyzed with Origin 8.5 (OriginLab, MA, USA).

Article Snippet: The integrase inhibition assay was determined with the HIV-1 Integrase Wild-Type Assay (XpressBio, Frederick, MD, USA) and the protease inhibitory assay was measured by using a SensoLyt™ 490 HIV-1 Protease Assay Kit * Fluorimetric * (ANASPEC).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Inhibition, Positive Control, Standard Deviation

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: Anti-HIV Activities and Mechanism of 12- O -Tricosanoylphorbol-20-acetate, a Novel Phorbol Ester from Ostodes katharinae

doi: 10.3390/molecules22091498

Figure Lengend Snippet: Hop-8 enhanced the expression of A3G and reduced p24 expression in HIV-1 producing cells and reduced the infectivity of the produced virus. ( A ) Hop-8 increased the expression of A3G and Vif while reduced the p24 expression in HIV-1 producing cells. The plasmids pcDNA3.1-APOBEC3G-HA (0.125 μg) and pNL4-3 (0.375 μg) were transfected into 2 × 10 5 293T cells. The compound was diluted to 6.75, 1.35, and 0.27 μM with DMEM and added to the wells 6 h post-transfection. The relative expression levels of A3G-HA, Vif, p55, and p24 were normalized by the levels of beta-actin. Values are presented as normalized intensities relative to the values of the pNL4-3 and A3G-HA positive groups (lane 8). Each data point represents the mean relative quantity ± standard deviation (bars), n ≥ 3. ( B ) Hop-8 reduced the infectivity of the progeny virus. The supernatant of NL4-3 was collected and 4 × 10 4 TZM-bl cells were infected with NL4-3 that contained 20 ng p24. The residual infection was determined using relative luciferase activity. All data represent the means ± standard deviation (bars), n ≥ 3. Data were analyzed by the unpair-sample t -test with Origin 8.5. ( p < 0.05, significant difference; *: p < 0.05; **: p < 0.01; NS: not significant). ( C ) Hop-8 increased the incorporation of A3G in progeny virions. pcDNA-APOBEC3G-HA (1 μg) and pNL4-3 (1 μg) were transfected consecutively or co-transfected into 293T cells in 6-well cell culture plates. Cells were cultured with or without hop-8 (2.7 μM) for 48 h. The supernatant was collected and centrifuged at 2000× g and the cell debris was discarded. The viral particles were lysed with 0.5% Triton-X100. A3G and p24 levels in the supernatant were determined by western blot. The relative expression levels of A3G-HA were normalized by the levels of p24. Each data point represents the mean relative quantity ± standard deviation (bars), n ≥ 3.

Article Snippet: The integrase inhibition assay was determined with the HIV-1 Integrase Wild-Type Assay (XpressBio, Frederick, MD, USA) and the protease inhibitory assay was measured by using a SensoLyt™ 490 HIV-1 Protease Assay Kit * Fluorimetric * (ANASPEC).

Techniques: Expressing, Infection, Produced, Transfection, Standard Deviation, Luciferase, Activity Assay, Cell Culture, Western Blot

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

Article Title: Anti-HIV Activities and Mechanism of 12- O -Tricosanoylphorbol-20-acetate, a Novel Phorbol Ester from Ostodes katharinae

doi: 10.3390/molecules22091498

Figure Lengend Snippet: Schematic of the anti-HIV-1 activity mechanism of hop-8. The process shown with deep green arrows represents a simple schematic of HIV replication. Hop-8 stimulates A3G expression via the PKC pathway. A3G is packaged into the progeny virions and restricts the replication of the progeny virions (shown with purple arrows).

Article Snippet: The integrase inhibition assay was determined with the HIV-1 Integrase Wild-Type Assay (XpressBio, Frederick, MD, USA) and the protease inhibitory assay was measured by using a SensoLyt™ 490 HIV-1 Protease Assay Kit * Fluorimetric * (ANASPEC).

Techniques: Activity Assay, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The Salmonella Type III Secretion Effector, Salmonella Leucine-rich Repeat Protein (SlrP), Targets the Human Chaperone ERdj3 *

doi: 10.1074/jbc.M110.100669

Figure Lengend Snippet: Bacterial strains, yeast strains, and plasmids used in this study

Article Snippet: E. coli DH5α supE44 Δ lacU 169 (Ø80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 ( 40 ) BL21 (DE3) F − ompT gal dcm lon hsdS B (r B − m B − ; E. coli B strain), with DE3, a λ prophage carrying the T7 RNA pol gene Stratagene S. enterica 14028 Wild type ATCC SV5193 14028 slrP ::3xFLAG, K m r ( 12 ) S. cerevisiae AH109 MATa, trp1–901, leu2–3, 112, ura3–52, his3–200, gal4 Δ, gal80 Δ, LYS2::GAL1 UAS -GAL1 TATA -HIS3, GAL2 UAS -GAL2 TATA -ADE2 URA3:: MEL1 UAS -MEL1 TATA -LacZ MEL1 A. Holtz, unpublished Y187 MAT α, ura3–52, his3–200, ade2–101, trp1–901, leu2–3, 112, gal4 Δ, gal80 Δ, met –, URA3:: GAL1 UAS -GAL1 TATA -LacZ MEL1 ( 41 ) Plasmids pcDNA3 Transient or stable transfection vector, Amp r Invitrogen pCS2+HA3 Transient transfection vector, Amp r F. Romero pGADT7 GAL4 (768–881) AD, LEU2 , Amp r Clontech pGBT10 GAL4 (1–147) DB, TRP1 , Amp r Clontech pGEX-4T-1 GST fusion vector, Amp r Pharmacia pGEX-4T-3 GST fusion vector, Amp r Pharmacia pIZ1623 pGEX-4T-3-SlrP ( 12 ) pIZ1627 pGBT10-SlrP ( 12 ) pIZ1706 pGADT7-ERdj3, clone obtained in the two-hybrid screen This study pIZ1708 pGEX-4T-1-ERdj3 This study pIZ1720 pCS2-SlrP-3xFLAG ( 12 ) pIZ1725 pcDNA3-SlrP-3xFLAG ( 12 ) pIZ1729 pCS2-ERdj3–3xHA This study pIZ1780 pcDNA3-SlrP(C546A)-3xFLAG ( 12 ) pIZ1791 pGADT7-ERdj3(23–358) This study pIZ1792 pGADT7-ERdj3(88–358) This study pIZ1793 pGADT7-ERdj3(129–358) This study pIZ1794 pGADT7-ERdj3(160–358) This study pIZ1795 pGADT7-ERdj3(201–358) This study pIZ1796 pGADT7-ERdj3(251–358) This study pIZ1797 pGEX-4T-1-ERdj3(23–358) This study pIZ1798 pGEX-4T-1-ERdj3(88–358) This study pIZ1799 pGEX-4T-1-ERdj3(129–358) This study pIZ1800 pGEX-4T-1-ERdj3(160–358) This study pIZ1801 pGEX-4T-1-ERdj3(201–358) This study pIZ1802 pGEX-4T-1-ERdj3(251–358) This study pIZ1804 pGBT10-SlrP(C546A) This study pQE10-BiP L. Hendershot Open in a separate window Bacterial strains, yeast strains, and plasmids used in this study

Techniques: Stable Transfection, Plasmid Preparation, Transfection, Two Hybrid Screening

Journal: The Journal of Biological Chemistry

Article Title: The Salmonella Type III Secretion Effector, Salmonella Leucine-rich Repeat Protein (SlrP), Targets the Human Chaperone ERdj3 *

doi: 10.1074/jbc.M110.100669

Figure Lengend Snippet: Interaction of Salmonella SlrP with human ERdj3 in the yeast two-hybrid system. A, diagram of the domains of full-length and deleted versions (1–6) of ERdj3. The domains of ERdj3 are indicated as: J, J domain; G/F, glycine-, and phenylalanine-rich domain; Ia, domain Ia; II, domain II; Ib, domain Ib; III, domain III. B, diploids were obtained by conjugation between strain AH109, containing derivatives of pGBT10 (DB-SlrP), and strain Y187, containing derivatives of pGADT7 (AD-ERdj3), as indicated. The interaction between the two hybrid proteins is shown by the growth in the absence of histidine and adenine. DB, fusion with the DNA-binding domain of Gal4; AD, fusion with the activation domain of Gal4; wt, wild-type SlrP; C546A, C546A mutant of SlrP; 1 to 6 correspond to ERdj3 subclones represented in A; −, empty vectors.

Article Snippet: E. coli DH5α supE44 Δ lacU 169 (Ø80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 ( 40 ) BL21 (DE3) F − ompT gal dcm lon hsdS B (r B − m B − ; E. coli B strain), with DE3, a λ prophage carrying the T7 RNA pol gene Stratagene S. enterica 14028 Wild type ATCC SV5193 14028 slrP ::3xFLAG, K m r ( 12 ) S. cerevisiae AH109 MATa, trp1–901, leu2–3, 112, ura3–52, his3–200, gal4 Δ, gal80 Δ, LYS2::GAL1 UAS -GAL1 TATA -HIS3, GAL2 UAS -GAL2 TATA -ADE2 URA3:: MEL1 UAS -MEL1 TATA -LacZ MEL1 A. Holtz, unpublished Y187 MAT α, ura3–52, his3–200, ade2–101, trp1–901, leu2–3, 112, gal4 Δ, gal80 Δ, met –, URA3:: GAL1 UAS -GAL1 TATA -LacZ MEL1 ( 41 ) Plasmids pcDNA3 Transient or stable transfection vector, Amp r Invitrogen pCS2+HA3 Transient transfection vector, Amp r F. Romero pGADT7 GAL4 (768–881) AD, LEU2 , Amp r Clontech pGBT10 GAL4 (1–147) DB, TRP1 , Amp r Clontech pGEX-4T-1 GST fusion vector, Amp r Pharmacia pGEX-4T-3 GST fusion vector, Amp r Pharmacia pIZ1623 pGEX-4T-3-SlrP ( 12 ) pIZ1627 pGBT10-SlrP ( 12 ) pIZ1706 pGADT7-ERdj3, clone obtained in the two-hybrid screen This study pIZ1708 pGEX-4T-1-ERdj3 This study pIZ1720 pCS2-SlrP-3xFLAG ( 12 ) pIZ1725 pcDNA3-SlrP-3xFLAG ( 12 ) pIZ1729 pCS2-ERdj3–3xHA This study pIZ1780 pcDNA3-SlrP(C546A)-3xFLAG ( 12 ) pIZ1791 pGADT7-ERdj3(23–358) This study pIZ1792 pGADT7-ERdj3(88–358) This study pIZ1793 pGADT7-ERdj3(129–358) This study pIZ1794 pGADT7-ERdj3(160–358) This study pIZ1795 pGADT7-ERdj3(201–358) This study pIZ1796 pGADT7-ERdj3(251–358) This study pIZ1797 pGEX-4T-1-ERdj3(23–358) This study pIZ1798 pGEX-4T-1-ERdj3(88–358) This study pIZ1799 pGEX-4T-1-ERdj3(129–358) This study pIZ1800 pGEX-4T-1-ERdj3(160–358) This study pIZ1801 pGEX-4T-1-ERdj3(201–358) This study pIZ1802 pGEX-4T-1-ERdj3(251–358) This study pIZ1804 pGBT10-SlrP(C546A) This study pQE10-BiP L. Hendershot Open in a separate window Bacterial strains, yeast strains, and plasmids used in this study

Techniques: Conjugation Assay, Binding Assay, Activation Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: The Salmonella Type III Secretion Effector, Salmonella Leucine-rich Repeat Protein (SlrP), Targets the Human Chaperone ERdj3 *

doi: 10.1074/jbc.M110.100669

Figure Lengend Snippet: Interaction of ERdj3 and SlrP in vitro. A, in vitro binding of SlrP-3×FLAG to GST-ERdj3. Expression of GST and GST-ERdj3 proteins was induced with isopropyl-1-thio-β-d-galactopyranoside, and proteins were isolated from bacterial lysates by affinity chromatography with glutathione-agarose beads. 4 μg of each GST protein were incubated with lysates from 109 cfu of S. enterica serovar Typhimurium strain SV5193 (14028 slrP::3×FLAG) prepared in Nonidet P-40 lysis buffer. After washing, proteins eluted in sample buffer were resolved in 12% SDS-PAGE, blotted on nitrocellulose filters, and developed with monoclonal anti-FLAG antibody. Lysate from 108 cfu of the same strain is also included for reference. B, domain II of ERdj3 is essential for interaction with SlrP. Deleted derivatives of ERdj3 1–6 (see Fig. 1A) were expressed in fusion with GST and used for binding experiments with lysates from 109 cfu of Salmonella strain SV5193 as in A. M, molecular mass markers: visible bands correspond to 150, 100, 75, 50, 37, and 25 kDa.

Article Snippet: E. coli DH5α supE44 Δ lacU 169 (Ø80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 ( 40 ) BL21 (DE3) F − ompT gal dcm lon hsdS B (r B − m B − ; E. coli B strain), with DE3, a λ prophage carrying the T7 RNA pol gene Stratagene S. enterica 14028 Wild type ATCC SV5193 14028 slrP ::3xFLAG, K m r ( 12 ) S. cerevisiae AH109 MATa, trp1–901, leu2–3, 112, ura3–52, his3–200, gal4 Δ, gal80 Δ, LYS2::GAL1 UAS -GAL1 TATA -HIS3, GAL2 UAS -GAL2 TATA -ADE2 URA3:: MEL1 UAS -MEL1 TATA -LacZ MEL1 A. Holtz, unpublished Y187 MAT α, ura3–52, his3–200, ade2–101, trp1–901, leu2–3, 112, gal4 Δ, gal80 Δ, met –, URA3:: GAL1 UAS -GAL1 TATA -LacZ MEL1 ( 41 ) Plasmids pcDNA3 Transient or stable transfection vector, Amp r Invitrogen pCS2+HA3 Transient transfection vector, Amp r F. Romero pGADT7 GAL4 (768–881) AD, LEU2 , Amp r Clontech pGBT10 GAL4 (1–147) DB, TRP1 , Amp r Clontech pGEX-4T-1 GST fusion vector, Amp r Pharmacia pGEX-4T-3 GST fusion vector, Amp r Pharmacia pIZ1623 pGEX-4T-3-SlrP ( 12 ) pIZ1627 pGBT10-SlrP ( 12 ) pIZ1706 pGADT7-ERdj3, clone obtained in the two-hybrid screen This study pIZ1708 pGEX-4T-1-ERdj3 This study pIZ1720 pCS2-SlrP-3xFLAG ( 12 ) pIZ1725 pcDNA3-SlrP-3xFLAG ( 12 ) pIZ1729 pCS2-ERdj3–3xHA This study pIZ1780 pcDNA3-SlrP(C546A)-3xFLAG ( 12 ) pIZ1791 pGADT7-ERdj3(23–358) This study pIZ1792 pGADT7-ERdj3(88–358) This study pIZ1793 pGADT7-ERdj3(129–358) This study pIZ1794 pGADT7-ERdj3(160–358) This study pIZ1795 pGADT7-ERdj3(201–358) This study pIZ1796 pGADT7-ERdj3(251–358) This study pIZ1797 pGEX-4T-1-ERdj3(23–358) This study pIZ1798 pGEX-4T-1-ERdj3(88–358) This study pIZ1799 pGEX-4T-1-ERdj3(129–358) This study pIZ1800 pGEX-4T-1-ERdj3(160–358) This study pIZ1801 pGEX-4T-1-ERdj3(201–358) This study pIZ1802 pGEX-4T-1-ERdj3(251–358) This study pIZ1804 pGBT10-SlrP(C546A) This study pQE10-BiP L. Hendershot Open in a separate window Bacterial strains, yeast strains, and plasmids used in this study

Techniques: In Vitro, Binding Assay, Expressing, Isolation, Affinity Chromatography, Incubation, Lysis, SDS Page

Journal: The Journal of Biological Chemistry

Article Title: The Salmonella Type III Secretion Effector, Salmonella Leucine-rich Repeat Protein (SlrP), Targets the Human Chaperone ERdj3 *

doi: 10.1074/jbc.M110.100669

Figure Lengend Snippet: Coimmunoprecipitation of SlrP and human ERdj3. HeLa cells were transiently co-transfected with 4 μg of two derivatives of plasmid pCS2, one expressing SlrP-3×FLAG, and the other ERdj3–3×HA (lane 1), or transfected with the plasmid expressing ERdj3–3×HA only (lane 2). Nonidet P-40 lysates from 5 × 106 transfected cells were subjected to immunoprecipitation (IP, right panels) with anti-FLAG monoclonal antibodies and, after the stringent washings described under “Experimental Procedures,” resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes, and developed with monoclonal anti-FLAG for the upper part of the membrane, and monoclonal anti-HA for the lower part. Lysates from 5 × 105 transfected cells are shown in the left panels.

Article Snippet: E. coli DH5α supE44 Δ lacU 169 (Ø80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 ( 40 ) BL21 (DE3) F − ompT gal dcm lon hsdS B (r B − m B − ; E. coli B strain), with DE3, a λ prophage carrying the T7 RNA pol gene Stratagene S. enterica 14028 Wild type ATCC SV5193 14028 slrP ::3xFLAG, K m r ( 12 ) S. cerevisiae AH109 MATa, trp1–901, leu2–3, 112, ura3–52, his3–200, gal4 Δ, gal80 Δ, LYS2::GAL1 UAS -GAL1 TATA -HIS3, GAL2 UAS -GAL2 TATA -ADE2 URA3:: MEL1 UAS -MEL1 TATA -LacZ MEL1 A. Holtz, unpublished Y187 MAT α, ura3–52, his3–200, ade2–101, trp1–901, leu2–3, 112, gal4 Δ, gal80 Δ, met –, URA3:: GAL1 UAS -GAL1 TATA -LacZ MEL1 ( 41 ) Plasmids pcDNA3 Transient or stable transfection vector, Amp r Invitrogen pCS2+HA3 Transient transfection vector, Amp r F. Romero pGADT7 GAL4 (768–881) AD, LEU2 , Amp r Clontech pGBT10 GAL4 (1–147) DB, TRP1 , Amp r Clontech pGEX-4T-1 GST fusion vector, Amp r Pharmacia pGEX-4T-3 GST fusion vector, Amp r Pharmacia pIZ1623 pGEX-4T-3-SlrP ( 12 ) pIZ1627 pGBT10-SlrP ( 12 ) pIZ1706 pGADT7-ERdj3, clone obtained in the two-hybrid screen This study pIZ1708 pGEX-4T-1-ERdj3 This study pIZ1720 pCS2-SlrP-3xFLAG ( 12 ) pIZ1725 pcDNA3-SlrP-3xFLAG ( 12 ) pIZ1729 pCS2-ERdj3–3xHA This study pIZ1780 pcDNA3-SlrP(C546A)-3xFLAG ( 12 ) pIZ1791 pGADT7-ERdj3(23–358) This study pIZ1792 pGADT7-ERdj3(88–358) This study pIZ1793 pGADT7-ERdj3(129–358) This study pIZ1794 pGADT7-ERdj3(160–358) This study pIZ1795 pGADT7-ERdj3(201–358) This study pIZ1796 pGADT7-ERdj3(251–358) This study pIZ1797 pGEX-4T-1-ERdj3(23–358) This study pIZ1798 pGEX-4T-1-ERdj3(88–358) This study pIZ1799 pGEX-4T-1-ERdj3(129–358) This study pIZ1800 pGEX-4T-1-ERdj3(160–358) This study pIZ1801 pGEX-4T-1-ERdj3(201–358) This study pIZ1802 pGEX-4T-1-ERdj3(251–358) This study pIZ1804 pGBT10-SlrP(C546A) This study pQE10-BiP L. Hendershot Open in a separate window Bacterial strains, yeast strains, and plasmids used in this study

Techniques: Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Bioprocessing, SDS Page, Membrane

Journal: The Journal of Biological Chemistry

Article Title: The Salmonella Type III Secretion Effector, Salmonella Leucine-rich Repeat Protein (SlrP), Targets the Human Chaperone ERdj3 *

doi: 10.1074/jbc.M110.100669

Figure Lengend Snippet: Colocalization of SlrP and ERdj3 in HeLa cells. A, microscopic analysis. HeLa cells transiently transfected with plasmids expressing SlrP-3×FLAG and ERdj3–3×HA were observed in a laser-scanning confocal microscope after permeabilization and staining with Alexa Fluor® 488 labeled anti-HA (green, upper panel) and Cy3-conjugated anti-FLAG (red, center panel). Superposition of the two labelings is shown in the lower panel. Yellowish staining indicates colocalization of SlrP and ERdj3 in the endoplasmic reticulum. Scale bar, 10 μm. B, detection of SlrP in the endoplasmic reticulum of HeLa-transfected cells. Total lysate (left panels) and microsomal fraction (right panels) from 107 HeLa cells stably expressing SlrP-3×FLAG were submitted to 15% SDS-PAGE, transferred to nitrocellulose membrane, and then probed with anti-FLAG monoclonal antibody to detect SlrP. The filter was also incubated with anti-ERdj3 and anti-Trx polyclonal antibodies to detect endogenous ERdj3, and endogenous Trx (a cytosolic and partially nuclear protein), respectively, as a control of the purity of the microsomal fraction. C, comparison of the migration of SlrP present in cytosolic and microsomal fractions of transfected HeLa cells. A cytosolic fraction (106 cells) and a microsomal fraction (107 cells) obtained from HeLa cells stably expressing SlrP-3×FLAG were submitted to 10% SDS-PAGE, transferred to nitrocellulose membrane and then probed with anti-FLAG monoclonal antibody to detect SlrP.

Article Snippet: E. coli DH5α supE44 Δ lacU 169 (Ø80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 ( 40 ) BL21 (DE3) F − ompT gal dcm lon hsdS B (r B − m B − ; E. coli B strain), with DE3, a λ prophage carrying the T7 RNA pol gene Stratagene S. enterica 14028 Wild type ATCC SV5193 14028 slrP ::3xFLAG, K m r ( 12 ) S. cerevisiae AH109 MATa, trp1–901, leu2–3, 112, ura3–52, his3–200, gal4 Δ, gal80 Δ, LYS2::GAL1 UAS -GAL1 TATA -HIS3, GAL2 UAS -GAL2 TATA -ADE2 URA3:: MEL1 UAS -MEL1 TATA -LacZ MEL1 A. Holtz, unpublished Y187 MAT α, ura3–52, his3–200, ade2–101, trp1–901, leu2–3, 112, gal4 Δ, gal80 Δ, met –, URA3:: GAL1 UAS -GAL1 TATA -LacZ MEL1 ( 41 ) Plasmids pcDNA3 Transient or stable transfection vector, Amp r Invitrogen pCS2+HA3 Transient transfection vector, Amp r F. Romero pGADT7 GAL4 (768–881) AD, LEU2 , Amp r Clontech pGBT10 GAL4 (1–147) DB, TRP1 , Amp r Clontech pGEX-4T-1 GST fusion vector, Amp r Pharmacia pGEX-4T-3 GST fusion vector, Amp r Pharmacia pIZ1623 pGEX-4T-3-SlrP ( 12 ) pIZ1627 pGBT10-SlrP ( 12 ) pIZ1706 pGADT7-ERdj3, clone obtained in the two-hybrid screen This study pIZ1708 pGEX-4T-1-ERdj3 This study pIZ1720 pCS2-SlrP-3xFLAG ( 12 ) pIZ1725 pcDNA3-SlrP-3xFLAG ( 12 ) pIZ1729 pCS2-ERdj3–3xHA This study pIZ1780 pcDNA3-SlrP(C546A)-3xFLAG ( 12 ) pIZ1791 pGADT7-ERdj3(23–358) This study pIZ1792 pGADT7-ERdj3(88–358) This study pIZ1793 pGADT7-ERdj3(129–358) This study pIZ1794 pGADT7-ERdj3(160–358) This study pIZ1795 pGADT7-ERdj3(201–358) This study pIZ1796 pGADT7-ERdj3(251–358) This study pIZ1797 pGEX-4T-1-ERdj3(23–358) This study pIZ1798 pGEX-4T-1-ERdj3(88–358) This study pIZ1799 pGEX-4T-1-ERdj3(129–358) This study pIZ1800 pGEX-4T-1-ERdj3(160–358) This study pIZ1801 pGEX-4T-1-ERdj3(201–358) This study pIZ1802 pGEX-4T-1-ERdj3(251–358) This study pIZ1804 pGBT10-SlrP(C546A) This study pQE10-BiP L. Hendershot Open in a separate window Bacterial strains, yeast strains, and plasmids used in this study

Techniques: Transfection, Expressing, Microscopy, Staining, Labeling, Stable Transfection, SDS Page, Membrane, Incubation, Control, Comparison, Migration

Journal: The Journal of Biological Chemistry

Article Title: The Salmonella Type III Secretion Effector, Salmonella Leucine-rich Repeat Protein (SlrP), Targets the Human Chaperone ERdj3 *

doi: 10.1074/jbc.M110.100669

Figure Lengend Snippet: Effect of SlrP on ERdj3 function. A, binding of ERdj3 to BiP. 50 μg of GST-ERdj3–1 (amino acids 23–358) or GST-ERdj3–3 (amino acids 129–358) immobilized in glutathione-agarose beads were incubated with 50 μg of His6-BiP in the presence or absence of 1 mm ATP and 50 μg of GST-SlrP as indicated. After incubating at 4 °C for 2 h, the beads were washed several times with binding buffer, and bound proteins were released by boiling for 5 min in sample buffer and subjected to 10% SDS-PAGE. Proteins were detected by Coomassie Blue staining. One stained gel is shown together with a graphical representation (below) of means ± S.D., from two independent experiments, of the quantification of bands of His6-Bip. B, stimulation of BiP ATPase activity by ERdj3. ATPase assays were performed with either no additions (None), 1 μg of His6-BiP plus 2.5 μg of GST (BiP), 1 μg of His6-BiP plus 2.5 μg of GST-ERdj3–1 plus 2.5 μg of GST (BiP+ERdj3), or 1 μg of His6-BiP plus 2.5 μg of GST-ERdj3–1 plus 2.5 μg GST-SlrP (BiP+ERdj3+SlrP). The amount of released phosphate was determined after incubation at 25 °C for 75 min with a non-radioactive procedure (see “Experimental Procedures”). Values are the mean of three separate experiments with an error bar representing S.D. C, binding of ERdj3 to dTg. Lysates from HeLa cells transiently expressing ERdj3–3×HA were mixed with lysates from untransfected cells (UTC) or with lysates transfected with SlrP or SlrPCys546Ala (C546A), as indicated, and then incubated with dTg or native protein A immobilized on agarose beads. After several washes with Nonidet P-40 buffer, proteins bound to the beads (Bi) were released by boiling in sample buffer, separated by using 10% SDS-PAGE and immunoblotted with anti-HA monoclonal antibodies. Part of the initial flow-through (F) obtained after the incubations was also included. D, image quantification of the relative amount of ERdj3 bound to immobilized dTg. The results are the mean ± S.D. of three independent experiments. The highest level was set to 100 for each experiment.

Article Snippet: E. coli DH5α supE44 Δ lacU 169 (Ø80 lacZ ΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 ( 40 ) BL21 (DE3) F − ompT gal dcm lon hsdS B (r B − m B − ; E. coli B strain), with DE3, a λ prophage carrying the T7 RNA pol gene Stratagene S. enterica 14028 Wild type ATCC SV5193 14028 slrP ::3xFLAG, K m r ( 12 ) S. cerevisiae AH109 MATa, trp1–901, leu2–3, 112, ura3–52, his3–200, gal4 Δ, gal80 Δ, LYS2::GAL1 UAS -GAL1 TATA -HIS3, GAL2 UAS -GAL2 TATA -ADE2 URA3:: MEL1 UAS -MEL1 TATA -LacZ MEL1 A. Holtz, unpublished Y187 MAT α, ura3–52, his3–200, ade2–101, trp1–901, leu2–3, 112, gal4 Δ, gal80 Δ, met –, URA3:: GAL1 UAS -GAL1 TATA -LacZ MEL1 ( 41 ) Plasmids pcDNA3 Transient or stable transfection vector, Amp r Invitrogen pCS2+HA3 Transient transfection vector, Amp r F. Romero pGADT7 GAL4 (768–881) AD, LEU2 , Amp r Clontech pGBT10 GAL4 (1–147) DB, TRP1 , Amp r Clontech pGEX-4T-1 GST fusion vector, Amp r Pharmacia pGEX-4T-3 GST fusion vector, Amp r Pharmacia pIZ1623 pGEX-4T-3-SlrP ( 12 ) pIZ1627 pGBT10-SlrP ( 12 ) pIZ1706 pGADT7-ERdj3, clone obtained in the two-hybrid screen This study pIZ1708 pGEX-4T-1-ERdj3 This study pIZ1720 pCS2-SlrP-3xFLAG ( 12 ) pIZ1725 pcDNA3-SlrP-3xFLAG ( 12 ) pIZ1729 pCS2-ERdj3–3xHA This study pIZ1780 pcDNA3-SlrP(C546A)-3xFLAG ( 12 ) pIZ1791 pGADT7-ERdj3(23–358) This study pIZ1792 pGADT7-ERdj3(88–358) This study pIZ1793 pGADT7-ERdj3(129–358) This study pIZ1794 pGADT7-ERdj3(160–358) This study pIZ1795 pGADT7-ERdj3(201–358) This study pIZ1796 pGADT7-ERdj3(251–358) This study pIZ1797 pGEX-4T-1-ERdj3(23–358) This study pIZ1798 pGEX-4T-1-ERdj3(88–358) This study pIZ1799 pGEX-4T-1-ERdj3(129–358) This study pIZ1800 pGEX-4T-1-ERdj3(160–358) This study pIZ1801 pGEX-4T-1-ERdj3(201–358) This study pIZ1802 pGEX-4T-1-ERdj3(251–358) This study pIZ1804 pGBT10-SlrP(C546A) This study pQE10-BiP L. Hendershot Open in a separate window Bacterial strains, yeast strains, and plasmids used in this study

Techniques: Binding Assay, Incubation, SDS Page, Staining, Activity Assay, Expressing, Transfection, Bioprocessing

Journal: Genome Biology

Article Title: Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression

doi: 10.1186/s13059-021-02304-3

Figure Lengend Snippet: HDR-mediated editing of Tspan2 CArG box. a Targeting strategy with CArG box sequence (red), protospacer sequence (green), PAM sequence (blue shade), and 3 bp substitution (blue) in ssODN repair template. b Allele-specific PCR of some founder mice with evidence of wild type (top) or mutant (bottom) edit and corresponding Sanger sequencing of CArG box (shaded). c qRT-PCR of Tspan2 in indicated tissues and genotypes ( n = 5–7 mice/genotype). Black, blue, and red bars here and below represent relative (wild type set to value of 1) mean Tspan2 mRNA (± STD) in wild type, heterozygous, and homozygous genotypes, respectively. d Average Ct values for indicated tissues ( n = 6–7 mice/tissue). Asterisks indicate p < 0.05. sg, sgRNA edited Tspan2 CArG box

Article Snippet: Fertilized oocytes derived from male B6(Cg)-Tyr 2J /J (Jackson Laboratory, stock #000058) and superovulated female FVB/NJ (Jackson Laboratory, stock #001800) mice were microinjected with 50 ng/μl of wild type Cas9 mRNA (TriLink Biotechnologies, San Diego, CA), 50 ng/μl of sgRNA (Synthego Corp., Menlo Park, CA), and 25 ng/μl of ULTRAmer standard desalting ssODN (Integrated DNA Technologies, Coralville, IA) harboring a CCT>GTC substitution in the Tspan2 CArG box (Fig. a).

Techniques: Sequencing, Mutagenesis, Quantitative RT-PCR

Journal: Genome Biology

Article Title: Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression

doi: 10.1186/s13059-021-02304-3

Figure Lengend Snippet: Spatial localization of Tspan2 mRNA in HDR-edited mouse tissues. Immunofluorescence (LMOD1) and RNA FISH ( Tspan2 ) of wild type aorta ( a ) and heart ( b ) versus Tspan2 sg/sg CArG box mutant (HDR) aorta ( c ) and heart ( d ). Arrows point to coronary vessels of the heart. Note decrease in Tspan2 mRNA (red dots) in vascular smooth muscle cells (labeled green with LMOD1 antibody) of aorta and coronary vessels in CArG box mutants ( c, d ). Scale bars are 50 μm. Representative images from n = 3 mice

Article Snippet: Fertilized oocytes derived from male B6(Cg)-Tyr 2J /J (Jackson Laboratory, stock #000058) and superovulated female FVB/NJ (Jackson Laboratory, stock #001800) mice were microinjected with 50 ng/μl of wild type Cas9 mRNA (TriLink Biotechnologies, San Diego, CA), 50 ng/μl of sgRNA (Synthego Corp., Menlo Park, CA), and 25 ng/μl of ULTRAmer standard desalting ssODN (Integrated DNA Technologies, Coralville, IA) harboring a CCT>GTC substitution in the Tspan2 CArG box (Fig. a).

Techniques: Immunofluorescence, Mutagenesis, Labeling

Journal: Genome Biology

Article Title: Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression

doi: 10.1186/s13059-021-02304-3

Figure Lengend Snippet: PE2-mediated editing of Tspan2 CArG box. a Targeting strategy with CArG box in red sequence, protospacer in green sequence, PAM in blue shaded sequence, and 1 bp substitution within RT template in blue. b Representative genotyping of several founders with correct installation of 1 bp transversion (left) and Sanger sequencing of CArG box showing correct edit in a mutant founder (right, shaded). c qRT-PCR of Tspan2 mRNA in indicated tissues and genotypes ( n = 4 mice per genotype). peg, pegRNA edited mouse; RT, reverse transcriptase; PBS, primer binding site

Article Snippet: Fertilized oocytes derived from male B6(Cg)-Tyr 2J /J (Jackson Laboratory, stock #000058) and superovulated female FVB/NJ (Jackson Laboratory, stock #001800) mice were microinjected with 50 ng/μl of wild type Cas9 mRNA (TriLink Biotechnologies, San Diego, CA), 50 ng/μl of sgRNA (Synthego Corp., Menlo Park, CA), and 25 ng/μl of ULTRAmer standard desalting ssODN (Integrated DNA Technologies, Coralville, IA) harboring a CCT>GTC substitution in the Tspan2 CArG box (Fig. a).

Techniques: Sequencing, Mutagenesis, Quantitative RT-PCR, Reverse Transcription, Binding Assay

Journal: Genome Biology

Article Title: Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression

doi: 10.1186/s13059-021-02304-3

Figure Lengend Snippet: Spatial localization of Tspan2 mRNA in PE2-edited mouse tissues. Immunofluorescence (LMOD1) and RNA FISH ( Tspan2 ) of blood vessels in wild type heart ( a ) and brain ( b ) versus Tspan2 peg/peg CArG box mutant heart ( c ) and brain ( d ). The Tspan2 mRNA is indicated by the red dots in vascular smooth muscle cells (labeled green with LMOD1 antibody) of wild type blood vessels ( a, b ), but is nearly absent in CArG mutant vessels ( c, d ). Scale bars are 50 μm. Representative images from n = 2 mice

Article Snippet: Fertilized oocytes derived from male B6(Cg)-Tyr 2J /J (Jackson Laboratory, stock #000058) and superovulated female FVB/NJ (Jackson Laboratory, stock #001800) mice were microinjected with 50 ng/μl of wild type Cas9 mRNA (TriLink Biotechnologies, San Diego, CA), 50 ng/μl of sgRNA (Synthego Corp., Menlo Park, CA), and 25 ng/μl of ULTRAmer standard desalting ssODN (Integrated DNA Technologies, Coralville, IA) harboring a CCT>GTC substitution in the Tspan2 CArG box (Fig. a).

Techniques: Immunofluorescence, Mutagenesis, Labeling

Journal: Genome Biology

Article Title: Prime editing in mice reveals the essentiality of a single base in driving tissue-specific gene expression

doi: 10.1186/s13059-021-02304-3

Figure Lengend Snippet: Genome-wide off-target analysis of HDR and PE2 edited founder mice. a Bar plot of number of CHANGE-seq sites detected using Cas9 WT and synthetic sgRNA or pegRNA targeting Tspan 2, on WT genomic DNA from same strain of mice used in HDR and PE2 editing experiments. b Venn diagram depicting common predicted off-target sites for sgRNA (orange) and pegRNA (blue) groups. c Manhattan plots of CHANGE-seq detected on- and off-target sites organized by chromosomal position, for sgRNA and pegRNA, with bar heights representing CHANGE-seq read count. Arrow indicates the on-target site. d, e Indel frequencies evaluated by rhAmpSeq at on- and off-target sites detected by CHANGE-seq for sgRNA founders ( d ) and for pegRNA founders ( e )

Article Snippet: Fertilized oocytes derived from male B6(Cg)-Tyr 2J /J (Jackson Laboratory, stock #000058) and superovulated female FVB/NJ (Jackson Laboratory, stock #001800) mice were microinjected with 50 ng/μl of wild type Cas9 mRNA (TriLink Biotechnologies, San Diego, CA), 50 ng/μl of sgRNA (Synthego Corp., Menlo Park, CA), and 25 ng/μl of ULTRAmer standard desalting ssODN (Integrated DNA Technologies, Coralville, IA) harboring a CCT>GTC substitution in the Tspan2 CArG box (Fig. a).

Techniques: Genome Wide