nucleosomes  (Worthington Biochemical)


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    Name:
    Carboxypeptidase B
    Description:
    Chromatographically purified A solution in 100 mM sodium chloride Chymotrypsin and trypsin ² 0 02
    Catalog Number:
    ls005301
    Price:
    140
    Size:
    10 mg
    Source:
    Porcine Pancreas
    Cas Number:
    9025.24.5
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    Structured Review

    Worthington Biochemical nucleosomes
    <t>Nucleosome</t> redistributions are determined by the underlying DNA sequence. ( A ) Western blots with the specified antibodies, at various times (in hours) after KSHV reactivation (hpr), of iSLK.219 cells treated with 0.2 µg/mL doxycycline. BRG1 protein
    Chromatographically purified A solution in 100 mM sodium chloride Chymotrypsin and trypsin ² 0 02
    https://www.bioz.com/result/nucleosomes/product/Worthington Biochemical
    Average 90 stars, based on 1008 article reviews
    Price from $9.99 to $1999.99
    nucleosomes - by Bioz Stars, 2020-10
    90/100 stars

    Images

    1) Product Images from "The spring-loaded genome: Nucleosome redistributions are widespread, transient, and DNA-directed"

    Article Title: The spring-loaded genome: Nucleosome redistributions are widespread, transient, and DNA-directed

    Journal: Genome Research

    doi: 10.1101/gr.160150.113

    Nucleosome redistributions are determined by the underlying DNA sequence. ( A ) Western blots with the specified antibodies, at various times (in hours) after KSHV reactivation (hpr), of iSLK.219 cells treated with 0.2 µg/mL doxycycline. BRG1 protein
    Figure Legend Snippet: Nucleosome redistributions are determined by the underlying DNA sequence. ( A ) Western blots with the specified antibodies, at various times (in hours) after KSHV reactivation (hpr), of iSLK.219 cells treated with 0.2 µg/mL doxycycline. BRG1 protein

    Techniques Used: Sequencing, Western Blot

    Reactivation of KSHV resulted in widespread, transient nucleosome redistribution. ( A ) Boxplot of the correlation values for the 472 loci nucleosome distributions between the 0-h time point and the time points following KSHV reactivation. ( B ) Number of
    Figure Legend Snippet: Reactivation of KSHV resulted in widespread, transient nucleosome redistribution. ( A ) Boxplot of the correlation values for the 472 loci nucleosome distributions between the 0-h time point and the time points following KSHV reactivation. ( B ) Number of

    Techniques Used:

    DNA sequence determined the concerted, widespread, transient redistribution of nucleosomes. ( A ) Average values for all genes identified as DNA-directed and DNA-independent, calculated by alignment of loci to the TSS for 0 h (black) and 24 h (red). ( B
    Figure Legend Snippet: DNA sequence determined the concerted, widespread, transient redistribution of nucleosomes. ( A ) Average values for all genes identified as DNA-directed and DNA-independent, calculated by alignment of loci to the TSS for 0 h (black) and 24 h (red). ( B

    Techniques Used: Sequencing

    Model of chromatin regulation in which nucleosome distributions move from a basal state architecture, to a transient intermediate state, then return to the basal architecture, in response to a common stimulus. The transient intermediate state's architecture
    Figure Legend Snippet: Model of chromatin regulation in which nucleosome distributions move from a basal state architecture, to a transient intermediate state, then return to the basal architecture, in response to a common stimulus. The transient intermediate state's architecture

    Techniques Used:

    2) Product Images from "Divergent Matrix-Remodeling Strategies Distinguish Developmental from Neoplastic Mammary Epithelial Cell Invasion Programs"

    Article Title: Divergent Matrix-Remodeling Strategies Distinguish Developmental from Neoplastic Mammary Epithelial Cell Invasion Programs

    Journal: Developmental cell

    doi: 10.1016/j.devcel.2018.08.025

    MT1-MMP Directs Mouse and Human Breast Carcinoma Invasion Programs Ex Vivo. (A) Bright-field micrographs of mammary carcinoma-derived organoids harvested from 3 month-old MMTV-PyMT +/− /Mmp14 f/f and MMTV-PyMT +/− /MMTV-Cre +/− /Mmp14 f/f mice, embedded in type I collagen hydrogels for 1 hour (left panels; scale bar, 50 μm); and MT1-MMP immunofluorescence with DAPI staining after a 4 day-culture period in collagen with FGF-2 (right panels) (scale bar, 20μm). Tumor organoid branch length is reduced from 93.5±4.3 μm with 5.7±0.3 branches/organoid in MMTV-PyMT +/− /Mmp14 f/f organoids (n=35) to 10.2±2.6 μm and 0.5±0.1 branches/organoid in MMTV-PyMT +/− /MMTV-Cre +/− /Mmp14 f/f organoids (n=28) (p
    Figure Legend Snippet: MT1-MMP Directs Mouse and Human Breast Carcinoma Invasion Programs Ex Vivo. (A) Bright-field micrographs of mammary carcinoma-derived organoids harvested from 3 month-old MMTV-PyMT +/− /Mmp14 f/f and MMTV-PyMT +/− /MMTV-Cre +/− /Mmp14 f/f mice, embedded in type I collagen hydrogels for 1 hour (left panels; scale bar, 50 μm); and MT1-MMP immunofluorescence with DAPI staining after a 4 day-culture period in collagen with FGF-2 (right panels) (scale bar, 20μm). Tumor organoid branch length is reduced from 93.5±4.3 μm with 5.7±0.3 branches/organoid in MMTV-PyMT +/− /Mmp14 f/f organoids (n=35) to 10.2±2.6 μm and 0.5±0.1 branches/organoid in MMTV-PyMT +/− /MMTV-Cre +/− /Mmp14 f/f organoids (n=28) (p

    Techniques Used: Ex Vivo, Derivative Assay, Mouse Assay, Immunofluorescence, Staining

    Mammary carcinoma-derived MT1-MMP dictates local invasion and metastasis. (A) LacZ staining of cross-sections from MMTV-PyMT +/− /Mmp14 +/LacZ (left panel) and MMTV-PyMT +/− /Mmp15 +/LacZ (right panel) mammary tumors with Eosin counterstaining (scale bar, 200 μm). (B-D) Kaplan-Meier plots depicting age of tumor onset (days) for (B) MMTV-PyMT +/− /Mmp14 f/f (n=40) versus MMTV-PyMT +/− /MMTV-Cre +/− /Mmp14 f/f (n=32) mice, (C) MMTV-PyMT +/− /Mmp15 f/f (n=12) versus MMTV-PyMT +/− /MMTV-Cre +/− /Mmp15 f/f (n=12) mice, and (D) MMTV-PyMT +/− /Mmp14 f/f /Mmp15 f/f (n=13) versus MMTV-PyMT +/− /MMTV-Cre +/− /Mmp14 f/f /Mmp15 f/f (n=9) mice. (E) 3-D Reconstructions of RFP fluorescence with phalloidin (F-Actin) and DAPI staining in mammary tumor cross-sections from 3–4 month-old MMTV-PyMT +/− /MMTV-Cre +/− /Rosa RFP and MMTV-PyMT +/− /MMTV-Cre +/− /Rosa RFP /Mmp14 f/f mice. Arrows mark strands of invasive tumor cells (scale bar, 50 μm). (F) Cytokeratin (CK)-8 and CK14 immunofluorescence with DAPI staining in mammary tumor cross-sections from 3–4 month old MMTV-PyMT +/− /Mmp14 f/f and MMTV-PyMT +/− /MMTV-Cre +/− /Mmp14 f/f mice (scale bar, 50 μm). A dotted line marks the tumor-stromal interface. (G) 3-D reconstructions of degraded collagen, labeled in situ with CF-CHP, in mammary tumors harvested from 3–4 month old MMTV-PyMT +/− /MMTV-Cre +/− /Rosa RFP and MMTV-PyMT +/− /MMTV-Cre +/− /Rosa RFP /Mmp14 f/f mice. Inset shows DAPI staining with dotted line marking the tumor-stromal interface and an asterisk within the tumor. Right panels show CF-CHP immunofluorescence together with RFP fluorescence (scale bar, 30 μm). CF-CHP immunofluorescence is reduced from 40.0±3.1 total pixels/μm 2 around MMTV-PyMT +/− /MMTV-Cre +/− /Rosa RFP tumors (n=8) to 10.0±1.9 total pixels/μm 2 around MMTV-PyMT +/− /MMTV-Cre +/− /Rosa RFP /Mmp14 f/f tumors (n=10), as normalized to scrambled CHP immunofluorescence (p
    Figure Legend Snippet: Mammary carcinoma-derived MT1-MMP dictates local invasion and metastasis. (A) LacZ staining of cross-sections from MMTV-PyMT +/− /Mmp14 +/LacZ (left panel) and MMTV-PyMT +/− /Mmp15 +/LacZ (right panel) mammary tumors with Eosin counterstaining (scale bar, 200 μm). (B-D) Kaplan-Meier plots depicting age of tumor onset (days) for (B) MMTV-PyMT +/− /Mmp14 f/f (n=40) versus MMTV-PyMT +/− /MMTV-Cre +/− /Mmp14 f/f (n=32) mice, (C) MMTV-PyMT +/− /Mmp15 f/f (n=12) versus MMTV-PyMT +/− /MMTV-Cre +/− /Mmp15 f/f (n=12) mice, and (D) MMTV-PyMT +/− /Mmp14 f/f /Mmp15 f/f (n=13) versus MMTV-PyMT +/− /MMTV-Cre +/− /Mmp14 f/f /Mmp15 f/f (n=9) mice. (E) 3-D Reconstructions of RFP fluorescence with phalloidin (F-Actin) and DAPI staining in mammary tumor cross-sections from 3–4 month-old MMTV-PyMT +/− /MMTV-Cre +/− /Rosa RFP and MMTV-PyMT +/− /MMTV-Cre +/− /Rosa RFP /Mmp14 f/f mice. Arrows mark strands of invasive tumor cells (scale bar, 50 μm). (F) Cytokeratin (CK)-8 and CK14 immunofluorescence with DAPI staining in mammary tumor cross-sections from 3–4 month old MMTV-PyMT +/− /Mmp14 f/f and MMTV-PyMT +/− /MMTV-Cre +/− /Mmp14 f/f mice (scale bar, 50 μm). A dotted line marks the tumor-stromal interface. (G) 3-D reconstructions of degraded collagen, labeled in situ with CF-CHP, in mammary tumors harvested from 3–4 month old MMTV-PyMT +/− /MMTV-Cre +/− /Rosa RFP and MMTV-PyMT +/− /MMTV-Cre +/− /Rosa RFP /Mmp14 f/f mice. Inset shows DAPI staining with dotted line marking the tumor-stromal interface and an asterisk within the tumor. Right panels show CF-CHP immunofluorescence together with RFP fluorescence (scale bar, 30 μm). CF-CHP immunofluorescence is reduced from 40.0±3.1 total pixels/μm 2 around MMTV-PyMT +/− /MMTV-Cre +/− /Rosa RFP tumors (n=8) to 10.0±1.9 total pixels/μm 2 around MMTV-PyMT +/− /MMTV-Cre +/− /Rosa RFP /Mmp14 f/f tumors (n=10), as normalized to scrambled CHP immunofluorescence (p

    Techniques Used: Derivative Assay, Staining, Mouse Assay, Fluorescence, Immunofluorescence, Labeling, In Situ

    3) Product Images from "TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp"

    Article Title: TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1704105114

    Models for TraR and the TraR–RNAP complex. ( A ) RaptorX-derived model for TraR. N- and C-termini are indicated, the four cysteine residues (C4) are tan spheres, and residues D3, D6, A8, I20, and E66 are in blue stick form. ( B ) TraR (blue) and DksA (green) were positioned manually based on alignment of TraR D3, D6, and A8 with DksA D71, D74, and A76, a portion of the TraR N-terminal α-helix, and the DksA α-helix 2 in its coiled-coil. Cysteines (C4) are yellow or tan spheres. ( C ) Model for TraR binding to E. coli ). The square corresponds to the area of the complex shown in expanded form in D and E . TraR is dark blue; the RNAP β-subunit is cyan, β′ is pink, the β′ secondary channel rim is yellow, and ω is pale blue. β′ Residues N680, K681A, and E677 are shown as orange spheres. ppGpp at site 1 is shown in red. TraR residues D3, D6, I44, A47, R48, I51, A47, and E66 are shown as blue spheres. ( D ) Enlarged view of TraR bound to RNAP secondary channel, as in C . I20 is in stick form. BH, bridge helix. ( E .
    Figure Legend Snippet: Models for TraR and the TraR–RNAP complex. ( A ) RaptorX-derived model for TraR. N- and C-termini are indicated, the four cysteine residues (C4) are tan spheres, and residues D3, D6, A8, I20, and E66 are in blue stick form. ( B ) TraR (blue) and DksA (green) were positioned manually based on alignment of TraR D3, D6, and A8 with DksA D71, D74, and A76, a portion of the TraR N-terminal α-helix, and the DksA α-helix 2 in its coiled-coil. Cysteines (C4) are yellow or tan spheres. ( C ) Model for TraR binding to E. coli ). The square corresponds to the area of the complex shown in expanded form in D and E . TraR is dark blue; the RNAP β-subunit is cyan, β′ is pink, the β′ secondary channel rim is yellow, and ω is pale blue. β′ Residues N680, K681A, and E677 are shown as orange spheres. ppGpp at site 1 is shown in red. TraR residues D3, D6, I44, A47, R48, I51, A47, and E66 are shown as blue spheres. ( D ) Enlarged view of TraR bound to RNAP secondary channel, as in C . I20 is in stick form. BH, bridge helix. ( E .

    Techniques Used: Derivative Assay, Binding Assay

    ( A ) Activation of p hisG , p livJ , or RNA-1 by TraR (0–500 nM). ( B ) Activation shown reflects maximum activation observed. Transcription of iraP P1 is activated to a different extent by TraR vs. DksA/ppGpp. Values plotted with TraR (1 µM), DksA (1 µM), or DksA (2 µM) and ppGpp (50 µM) together, relative to transcription without factors (set to 1).
    Figure Legend Snippet: ( A ) Activation of p hisG , p livJ , or RNA-1 by TraR (0–500 nM). ( B ) Activation shown reflects maximum activation observed. Transcription of iraP P1 is activated to a different extent by TraR vs. DksA/ppGpp. Values plotted with TraR (1 µM), DksA (1 µM), or DksA (2 µM) and ppGpp (50 µM) together, relative to transcription without factors (set to 1).

    Techniques Used: Activation Assay

    A half-DksA variant, similar in length to WT TraR, does not complement a ∆ dksA mutant. ( A ) Representative Western blot from cell lysates made from a ∆ dksA strain carrying either the WT gene or the half - dksA gene fused to an IPTG-inducible promoter. Cells were harvested 1, 2, or 3 h after induction with 0.5 mM IPTG. One-microgram of cell lysate was loaded in each lane, and purified DksA-HMK was loaded in lane 1 for comparison. Somewhat lower amounts of the half-DksA variant were observed than of WT DksA, which could be attributable to lower stability of the half-DksA variant or to reduced ability of the DksA antibody to recognize the half-DksA peptide. ( B and C ) Growth on plates containing defined medium without amino acids and ( B ) 0.1 mM IPTG or ( C ) 1 mM IPTG. Sector 1: empty vector control. Sector 2: plasmid containing half - dksA gene. Sector 3: plasmid containing the full-length traR gene. Sector 4: plasmid containing the full-length dksA gene. Even if the half-DksA concentration was lower than the WT DksA concentration, it is likely that the half-DksA would have still resulted in at least partial complementation, because we showed previously that even 50% of the WT concentration supplied from a plasmid was sufficient to complement a ∆ dksA ).
    Figure Legend Snippet: A half-DksA variant, similar in length to WT TraR, does not complement a ∆ dksA mutant. ( A ) Representative Western blot from cell lysates made from a ∆ dksA strain carrying either the WT gene or the half - dksA gene fused to an IPTG-inducible promoter. Cells were harvested 1, 2, or 3 h after induction with 0.5 mM IPTG. One-microgram of cell lysate was loaded in each lane, and purified DksA-HMK was loaded in lane 1 for comparison. Somewhat lower amounts of the half-DksA variant were observed than of WT DksA, which could be attributable to lower stability of the half-DksA variant or to reduced ability of the DksA antibody to recognize the half-DksA peptide. ( B and C ) Growth on plates containing defined medium without amino acids and ( B ) 0.1 mM IPTG or ( C ) 1 mM IPTG. Sector 1: empty vector control. Sector 2: plasmid containing half - dksA gene. Sector 3: plasmid containing the full-length traR gene. Sector 4: plasmid containing the full-length dksA gene. Even if the half-DksA concentration was lower than the WT DksA concentration, it is likely that the half-DksA would have still resulted in at least partial complementation, because we showed previously that even 50% of the WT concentration supplied from a plasmid was sufficient to complement a ∆ dksA ).

    Techniques Used: Variant Assay, Mutagenesis, Western Blot, Purification, Plasmid Preparation, Concentration Assay

    TraR is more active than DksA for inhibition of transcription but has a similar affinity for RNAP. ( A ) Multiround in vitro transcription of rrnB P1 or lacUV 5 at a range of concentrations of TraR (wedge indicates 1 nM to 1 µM for rrnB P1 or 1 nM to 2 µM for lacUV5 ) or of DksA (wedge indicates 4 nM to 8 µM). Plasmid templates also contained the RNA-1 promoter. ( B ) Quantification of transcripts from experiments like those in A plotted relative to values in the absence of TraR or DksA. The IC 50 for inhibition by TraR was ∼50 nM and for DksA ∼1.3 µM [averages with SDs from at least three independent experiments ( n = 3)]. ( C ) Cross-linking with β′ R933-Bpa RNAP, β′ Q929-Bpa RNAP, or β′ R1148-Bpa RNAP with 32 P-TraR or 32 P-DksA. The portion of a representative 4–12% SDS gel containing the cross-linked β′-DksA or β′-TraR products is shown. ( D ) Unlabeled DksA or TraR competes similarly for binding of 32 P-labeled HMK-DksA to RNAP. Unlabeled DksA or TraR (0–16 µM) was added to 1 µM 32 P-DksA and 0.1 µM core RNAP before Fe 2+ -mediated cleavage of DksA. Fraction of 32 P-DksA cleaved was normalized to that in the absence of competitor. Next, 1 µM unlabeled DksA or 0.6 µM unlabeled TraR reduced cleavage of 1 µM 32 P-DksA by ∼50% ( n = 3). ( E ) Representative gel showing DNase I footprints of RNAP bound to the rrnB P1 promoter, 3′ end-labeled on the template strand, with or without TraR or DksA. DNase I digested fragment without RNAP or added factors (lanes 1 and 2), with RNAP alone (lanes 3 and 4), with RNAP + 5 µM DksA (lanes 5 and 6), or with RNAP and 5 µM TraR (lanes 7 and 8). Undigested fragment (lane 9). A+G sequence ladder is on the Left . Traces of gel lanes showing extent of protection are on the Right . Colored dots indicate the downstream boundary of DNase I protection without (green dot; ∼+12), or with (red or blue dots; ∼+1) DksA or TraR. The upstream boundary of protection in lanes 3–8 is ∼−59 ( n = 3). ( F ) TraR and DksA alter the lifetime of rrnB P1(dis) promoter complexes in vitro. RNAP–promoter complexes were preformed with TraR (15 nM) or DksA (15 nM or 500 nM), or without factors, and the fraction remaining at the indicated times after heparin addition was determined by transcription. Half-lives of rrnB P1(dis) complexes: no added factor, 18 min; 15 nM TraR, 3 min; 15 nM DksA, 18 min; 500 nM DksA, 6 min. Error bars indicate the range from two independent experiments ( n = 2).
    Figure Legend Snippet: TraR is more active than DksA for inhibition of transcription but has a similar affinity for RNAP. ( A ) Multiround in vitro transcription of rrnB P1 or lacUV 5 at a range of concentrations of TraR (wedge indicates 1 nM to 1 µM for rrnB P1 or 1 nM to 2 µM for lacUV5 ) or of DksA (wedge indicates 4 nM to 8 µM). Plasmid templates also contained the RNA-1 promoter. ( B ) Quantification of transcripts from experiments like those in A plotted relative to values in the absence of TraR or DksA. The IC 50 for inhibition by TraR was ∼50 nM and for DksA ∼1.3 µM [averages with SDs from at least three independent experiments ( n = 3)]. ( C ) Cross-linking with β′ R933-Bpa RNAP, β′ Q929-Bpa RNAP, or β′ R1148-Bpa RNAP with 32 P-TraR or 32 P-DksA. The portion of a representative 4–12% SDS gel containing the cross-linked β′-DksA or β′-TraR products is shown. ( D ) Unlabeled DksA or TraR competes similarly for binding of 32 P-labeled HMK-DksA to RNAP. Unlabeled DksA or TraR (0–16 µM) was added to 1 µM 32 P-DksA and 0.1 µM core RNAP before Fe 2+ -mediated cleavage of DksA. Fraction of 32 P-DksA cleaved was normalized to that in the absence of competitor. Next, 1 µM unlabeled DksA or 0.6 µM unlabeled TraR reduced cleavage of 1 µM 32 P-DksA by ∼50% ( n = 3). ( E ) Representative gel showing DNase I footprints of RNAP bound to the rrnB P1 promoter, 3′ end-labeled on the template strand, with or without TraR or DksA. DNase I digested fragment without RNAP or added factors (lanes 1 and 2), with RNAP alone (lanes 3 and 4), with RNAP + 5 µM DksA (lanes 5 and 6), or with RNAP and 5 µM TraR (lanes 7 and 8). Undigested fragment (lane 9). A+G sequence ladder is on the Left . Traces of gel lanes showing extent of protection are on the Right . Colored dots indicate the downstream boundary of DNase I protection without (green dot; ∼+12), or with (red or blue dots; ∼+1) DksA or TraR. The upstream boundary of protection in lanes 3–8 is ∼−59 ( n = 3). ( F ) TraR and DksA alter the lifetime of rrnB P1(dis) promoter complexes in vitro. RNAP–promoter complexes were preformed with TraR (15 nM) or DksA (15 nM or 500 nM), or without factors, and the fraction remaining at the indicated times after heparin addition was determined by transcription. Half-lives of rrnB P1(dis) complexes: no added factor, 18 min; 15 nM TraR, 3 min; 15 nM DksA, 18 min; 500 nM DksA, 6 min. Error bars indicate the range from two independent experiments ( n = 2).

    Techniques Used: Inhibition, In Vitro, Plasmid Preparation, SDS-Gel, Binding Assay, Labeling, Sequencing

    4) Product Images from "TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp"

    Article Title: TraR directly regulates transcription initiation by mimicking the combined effects of the global regulators DksA and ppGpp

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1704105114

    Models for TraR and the TraR–RNAP complex. ( A ) RaptorX-derived model for TraR. N- and C-termini are indicated, the four cysteine residues (C4) are tan spheres, and residues D3, D6, A8, I20, and E66 are in blue stick form. ( B ) TraR (blue) and DksA (green) were positioned manually based on alignment of TraR D3, D6, and A8 with DksA D71, D74, and A76, a portion of the TraR N-terminal α-helix, and the DksA α-helix 2 in its coiled-coil. Cysteines (C4) are yellow or tan spheres. ( C ) Model for TraR binding to E. coli ). The square corresponds to the area of the complex shown in expanded form in D and E . TraR is dark blue; the RNAP β-subunit is cyan, β′ is pink, the β′ secondary channel rim is yellow, and ω is pale blue. β′ Residues N680, K681A, and E677 are shown as orange spheres. ppGpp at site 1 is shown in red. TraR residues D3, D6, I44, A47, R48, I51, A47, and E66 are shown as blue spheres. ( D ) Enlarged view of TraR bound to RNAP secondary channel, as in C . I20 is in stick form. BH, bridge helix. ( E .
    Figure Legend Snippet: Models for TraR and the TraR–RNAP complex. ( A ) RaptorX-derived model for TraR. N- and C-termini are indicated, the four cysteine residues (C4) are tan spheres, and residues D3, D6, A8, I20, and E66 are in blue stick form. ( B ) TraR (blue) and DksA (green) were positioned manually based on alignment of TraR D3, D6, and A8 with DksA D71, D74, and A76, a portion of the TraR N-terminal α-helix, and the DksA α-helix 2 in its coiled-coil. Cysteines (C4) are yellow or tan spheres. ( C ) Model for TraR binding to E. coli ). The square corresponds to the area of the complex shown in expanded form in D and E . TraR is dark blue; the RNAP β-subunit is cyan, β′ is pink, the β′ secondary channel rim is yellow, and ω is pale blue. β′ Residues N680, K681A, and E677 are shown as orange spheres. ppGpp at site 1 is shown in red. TraR residues D3, D6, I44, A47, R48, I51, A47, and E66 are shown as blue spheres. ( D ) Enlarged view of TraR bound to RNAP secondary channel, as in C . I20 is in stick form. BH, bridge helix. ( E .

    Techniques Used: Derivative Assay, Binding Assay

    ( A ) Activation of p hisG , p livJ , or RNA-1 by TraR (0–500 nM). ( B ) Activation shown reflects maximum activation observed. Transcription of iraP P1 is activated to a different extent by TraR vs. DksA/ppGpp. Values plotted with TraR (1 µM), DksA (1 µM), or DksA (2 µM) and ppGpp (50 µM) together, relative to transcription without factors (set to 1).
    Figure Legend Snippet: ( A ) Activation of p hisG , p livJ , or RNA-1 by TraR (0–500 nM). ( B ) Activation shown reflects maximum activation observed. Transcription of iraP P1 is activated to a different extent by TraR vs. DksA/ppGpp. Values plotted with TraR (1 µM), DksA (1 µM), or DksA (2 µM) and ppGpp (50 µM) together, relative to transcription without factors (set to 1).

    Techniques Used: Activation Assay

    A half-DksA variant, similar in length to WT TraR, does not complement a ∆ dksA mutant. ( A ) Representative Western blot from cell lysates made from a ∆ dksA strain carrying either the WT gene or the half - dksA gene fused to an IPTG-inducible promoter. Cells were harvested 1, 2, or 3 h after induction with 0.5 mM IPTG. One-microgram of cell lysate was loaded in each lane, and purified DksA-HMK was loaded in lane 1 for comparison. Somewhat lower amounts of the half-DksA variant were observed than of WT DksA, which could be attributable to lower stability of the half-DksA variant or to reduced ability of the DksA antibody to recognize the half-DksA peptide. ( B and C ) Growth on plates containing defined medium without amino acids and ( B ) 0.1 mM IPTG or ( C ) 1 mM IPTG. Sector 1: empty vector control. Sector 2: plasmid containing half - dksA gene. Sector 3: plasmid containing the full-length traR gene. Sector 4: plasmid containing the full-length dksA gene. Even if the half-DksA concentration was lower than the WT DksA concentration, it is likely that the half-DksA would have still resulted in at least partial complementation, because we showed previously that even 50% of the WT concentration supplied from a plasmid was sufficient to complement a ∆ dksA ).
    Figure Legend Snippet: A half-DksA variant, similar in length to WT TraR, does not complement a ∆ dksA mutant. ( A ) Representative Western blot from cell lysates made from a ∆ dksA strain carrying either the WT gene or the half - dksA gene fused to an IPTG-inducible promoter. Cells were harvested 1, 2, or 3 h after induction with 0.5 mM IPTG. One-microgram of cell lysate was loaded in each lane, and purified DksA-HMK was loaded in lane 1 for comparison. Somewhat lower amounts of the half-DksA variant were observed than of WT DksA, which could be attributable to lower stability of the half-DksA variant or to reduced ability of the DksA antibody to recognize the half-DksA peptide. ( B and C ) Growth on plates containing defined medium without amino acids and ( B ) 0.1 mM IPTG or ( C ) 1 mM IPTG. Sector 1: empty vector control. Sector 2: plasmid containing half - dksA gene. Sector 3: plasmid containing the full-length traR gene. Sector 4: plasmid containing the full-length dksA gene. Even if the half-DksA concentration was lower than the WT DksA concentration, it is likely that the half-DksA would have still resulted in at least partial complementation, because we showed previously that even 50% of the WT concentration supplied from a plasmid was sufficient to complement a ∆ dksA ).

    Techniques Used: Variant Assay, Mutagenesis, Western Blot, Purification, Plasmid Preparation, Concentration Assay

    TraR is more active than DksA for inhibition of transcription but has a similar affinity for RNAP. ( A ) Multiround in vitro transcription of rrnB P1 or lacUV 5 at a range of concentrations of TraR (wedge indicates 1 nM to 1 µM for rrnB P1 or 1 nM to 2 µM for lacUV5 ) or of DksA (wedge indicates 4 nM to 8 µM). Plasmid templates also contained the RNA-1 promoter. ( B ) Quantification of transcripts from experiments like those in A plotted relative to values in the absence of TraR or DksA. The IC 50 for inhibition by TraR was ∼50 nM and for DksA ∼1.3 µM [averages with SDs from at least three independent experiments ( n = 3)]. ( C ) Cross-linking with β′ R933-Bpa RNAP, β′ Q929-Bpa RNAP, or β′ R1148-Bpa RNAP with 32 P-TraR or 32 P-DksA. The portion of a representative 4–12% SDS gel containing the cross-linked β′-DksA or β′-TraR products is shown. ( D ) Unlabeled DksA or TraR competes similarly for binding of 32 P-labeled HMK-DksA to RNAP. Unlabeled DksA or TraR (0–16 µM) was added to 1 µM 32 P-DksA and 0.1 µM core RNAP before Fe 2+ -mediated cleavage of DksA. Fraction of 32 P-DksA cleaved was normalized to that in the absence of competitor. Next, 1 µM unlabeled DksA or 0.6 µM unlabeled TraR reduced cleavage of 1 µM 32 P-DksA by ∼50% ( n = 3). ( E ) Representative gel showing DNase I footprints of RNAP bound to the rrnB P1 promoter, 3′ end-labeled on the template strand, with or without TraR or DksA. DNase I digested fragment without RNAP or added factors (lanes 1 and 2), with RNAP alone (lanes 3 and 4), with RNAP + 5 µM DksA (lanes 5 and 6), or with RNAP and 5 µM TraR (lanes 7 and 8). Undigested fragment (lane 9). A+G sequence ladder is on the Left . Traces of gel lanes showing extent of protection are on the Right . Colored dots indicate the downstream boundary of DNase I protection without (green dot; ∼+12), or with (red or blue dots; ∼+1) DksA or TraR. The upstream boundary of protection in lanes 3–8 is ∼−59 ( n = 3). ( F ) TraR and DksA alter the lifetime of rrnB P1(dis) promoter complexes in vitro. RNAP–promoter complexes were preformed with TraR (15 nM) or DksA (15 nM or 500 nM), or without factors, and the fraction remaining at the indicated times after heparin addition was determined by transcription. Half-lives of rrnB P1(dis) complexes: no added factor, 18 min; 15 nM TraR, 3 min; 15 nM DksA, 18 min; 500 nM DksA, 6 min. Error bars indicate the range from two independent experiments ( n = 2).
    Figure Legend Snippet: TraR is more active than DksA for inhibition of transcription but has a similar affinity for RNAP. ( A ) Multiround in vitro transcription of rrnB P1 or lacUV 5 at a range of concentrations of TraR (wedge indicates 1 nM to 1 µM for rrnB P1 or 1 nM to 2 µM for lacUV5 ) or of DksA (wedge indicates 4 nM to 8 µM). Plasmid templates also contained the RNA-1 promoter. ( B ) Quantification of transcripts from experiments like those in A plotted relative to values in the absence of TraR or DksA. The IC 50 for inhibition by TraR was ∼50 nM and for DksA ∼1.3 µM [averages with SDs from at least three independent experiments ( n = 3)]. ( C ) Cross-linking with β′ R933-Bpa RNAP, β′ Q929-Bpa RNAP, or β′ R1148-Bpa RNAP with 32 P-TraR or 32 P-DksA. The portion of a representative 4–12% SDS gel containing the cross-linked β′-DksA or β′-TraR products is shown. ( D ) Unlabeled DksA or TraR competes similarly for binding of 32 P-labeled HMK-DksA to RNAP. Unlabeled DksA or TraR (0–16 µM) was added to 1 µM 32 P-DksA and 0.1 µM core RNAP before Fe 2+ -mediated cleavage of DksA. Fraction of 32 P-DksA cleaved was normalized to that in the absence of competitor. Next, 1 µM unlabeled DksA or 0.6 µM unlabeled TraR reduced cleavage of 1 µM 32 P-DksA by ∼50% ( n = 3). ( E ) Representative gel showing DNase I footprints of RNAP bound to the rrnB P1 promoter, 3′ end-labeled on the template strand, with or without TraR or DksA. DNase I digested fragment without RNAP or added factors (lanes 1 and 2), with RNAP alone (lanes 3 and 4), with RNAP + 5 µM DksA (lanes 5 and 6), or with RNAP and 5 µM TraR (lanes 7 and 8). Undigested fragment (lane 9). A+G sequence ladder is on the Left . Traces of gel lanes showing extent of protection are on the Right . Colored dots indicate the downstream boundary of DNase I protection without (green dot; ∼+12), or with (red or blue dots; ∼+1) DksA or TraR. The upstream boundary of protection in lanes 3–8 is ∼−59 ( n = 3). ( F ) TraR and DksA alter the lifetime of rrnB P1(dis) promoter complexes in vitro. RNAP–promoter complexes were preformed with TraR (15 nM) or DksA (15 nM or 500 nM), or without factors, and the fraction remaining at the indicated times after heparin addition was determined by transcription. Half-lives of rrnB P1(dis) complexes: no added factor, 18 min; 15 nM TraR, 3 min; 15 nM DksA, 18 min; 500 nM DksA, 6 min. Error bars indicate the range from two independent experiments ( n = 2).

    Techniques Used: Inhibition, In Vitro, Plasmid Preparation, SDS-Gel, Binding Assay, Labeling, Sequencing

    5) Product Images from "Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment"

    Article Title: Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment

    Journal: Human Vaccines & Immunotherapeutics

    doi: 10.4161/21645515.2014.990856

    High performance strong cation exchange chromatography of the h2E2 antibody before and after treatment with carboxypeptidase B. 100 μg of h2E2 antibody was injected, and eluted with a gradient of NaCl in MES buffer. Note the disappearance of peaks 1 and 2 after removal of the C-terminal lysine residues. These peaks represent h2E2 antibody molecules containing 1 or 2 lysine residues on the C-termini of the 2 heavy chains in the antibody (which, after removal of lysine by carboxypeptidase elute with the main peak labeled ‘0” eluting at approximately 11 minutes).
    Figure Legend Snippet: High performance strong cation exchange chromatography of the h2E2 antibody before and after treatment with carboxypeptidase B. 100 μg of h2E2 antibody was injected, and eluted with a gradient of NaCl in MES buffer. Note the disappearance of peaks 1 and 2 after removal of the C-terminal lysine residues. These peaks represent h2E2 antibody molecules containing 1 or 2 lysine residues on the C-termini of the 2 heavy chains in the antibody (which, after removal of lysine by carboxypeptidase elute with the main peak labeled ‘0” eluting at approximately 11 minutes).

    Techniques Used: Chromatography, Injection, Labeling

    6) Product Images from "Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing"

    Article Title: Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing

    Journal: RNA Biology

    doi: 10.1080/15476286.2018.1462654

    Reverse transcription through CMC-modified Ψ. (A) Chemical structures of Ψ and CMC-modified Ψ (CMC-Ψ). (B) Reverse transcription of a synthetic RNA oligo (Oligo Ψa) containing Ψ or CMC-Ψ with AMV RT or HIV RT. RNA oligo sequence: 5′- UACACUCAGXUCGGACUAAAGCUGCUC (X = Ψ or CMC-Ψ). (C) Quantification of Ψ or CMC-Ψ read-through by different reverse transcriptase enzymes under varying divalent cation conditions.
    Figure Legend Snippet: Reverse transcription through CMC-modified Ψ. (A) Chemical structures of Ψ and CMC-modified Ψ (CMC-Ψ). (B) Reverse transcription of a synthetic RNA oligo (Oligo Ψa) containing Ψ or CMC-Ψ with AMV RT or HIV RT. RNA oligo sequence: 5′- UACACUCAGXUCGGACUAAAGCUGCUC (X = Ψ or CMC-Ψ). (C) Quantification of Ψ or CMC-Ψ read-through by different reverse transcriptase enzymes under varying divalent cation conditions.

    Techniques Used: Modification, Sequencing

    7) Product Images from "Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment"

    Article Title: Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment

    Journal: Human Vaccines & Immunotherapeutics

    doi: 10.4161/21645515.2014.990856

    10% SDS-PAGE gel analysis of TBP reduced and ABD-F labeled monoclonal antibody h2E2 heavy and light chains. 0.2 mg/ml antibody was first reduced with 4 mM TBP at 60°C for the times indicated in the figure, followed by alkylation with 4 mM ABD-F for 15 minutes at 22°C. Aliquots (5 μg) of the resultant samples were diluted in SDS-PAGE sample buffer and run on a 10% gel. Following photography under UV light to detect incorporated ABD-cys fluorescence (left hand side); the gel was then stained to measure total protein, and re-photographed (right hand side). Migratory positions of the heavy and light chains are indicated.
    Figure Legend Snippet: 10% SDS-PAGE gel analysis of TBP reduced and ABD-F labeled monoclonal antibody h2E2 heavy and light chains. 0.2 mg/ml antibody was first reduced with 4 mM TBP at 60°C for the times indicated in the figure, followed by alkylation with 4 mM ABD-F for 15 minutes at 22°C. Aliquots (5 μg) of the resultant samples were diluted in SDS-PAGE sample buffer and run on a 10% gel. Following photography under UV light to detect incorporated ABD-cys fluorescence (left hand side); the gel was then stained to measure total protein, and re-photographed (right hand side). Migratory positions of the heavy and light chains are indicated.

    Techniques Used: SDS Page, Labeling, Fluorescence, Staining

    High performance strong cation exchange chromatography of the Fab fragment derived from the h2E2 antibody. 100 μg purified Fab fragment was injected, and eluted with a gradient of NaCl in MES buffer at 22°C. Note the relative lack of charge heterogeneity compared to the intact h2E2 antibody ( Fig. 4 and the inset), indicating much of the charge heterogeneity resides in the Fc portion of the h2E2 antibody. For comparative purposes, the position and pattern of the intact antibody on the same column using the same buffers and gradient is shown in the inset (offset in the y-direction for clarity).
    Figure Legend Snippet: High performance strong cation exchange chromatography of the Fab fragment derived from the h2E2 antibody. 100 μg purified Fab fragment was injected, and eluted with a gradient of NaCl in MES buffer at 22°C. Note the relative lack of charge heterogeneity compared to the intact h2E2 antibody ( Fig. 4 and the inset), indicating much of the charge heterogeneity resides in the Fc portion of the h2E2 antibody. For comparative purposes, the position and pattern of the intact antibody on the same column using the same buffers and gradient is shown in the inset (offset in the y-direction for clarity).

    Techniques Used: Chromatography, Derivative Assay, Purification, Injection

    High performance strong cation exchange chromatography of the h2E2 antibody before and after treatment with carboxypeptidase B. 100 μg of h2E2 antibody was injected, and eluted with a gradient of NaCl in MES buffer. Note the disappearance of peaks 1 and 2 after removal of the C-terminal lysine residues. These peaks represent h2E2 antibody molecules containing 1 or 2 lysine residues on the C-termini of the 2 heavy chains in the antibody (which, after removal of lysine by carboxypeptidase elute with the main peak labeled ‘0” eluting at approximately 11 minutes).
    Figure Legend Snippet: High performance strong cation exchange chromatography of the h2E2 antibody before and after treatment with carboxypeptidase B. 100 μg of h2E2 antibody was injected, and eluted with a gradient of NaCl in MES buffer. Note the disappearance of peaks 1 and 2 after removal of the C-terminal lysine residues. These peaks represent h2E2 antibody molecules containing 1 or 2 lysine residues on the C-termini of the 2 heavy chains in the antibody (which, after removal of lysine by carboxypeptidase elute with the main peak labeled ‘0” eluting at approximately 11 minutes).

    Techniques Used: Chromatography, Injection, Labeling

    Intrinsic h2E2 antibody tryptophan and tyrosine fluorescence quenching by cocaine binding. Shown are emission spectra of 5 nM h2E2 antibody both before and after addition of 100 nM cocaine, with excitation at either 280 nm (tyrosine and tryptophan) or 295 nm (tryptophan). Note the decrease in emission (quenching) caused by cocaine, but little if any change in the emission maximum (near 330 nm in both cases).
    Figure Legend Snippet: Intrinsic h2E2 antibody tryptophan and tyrosine fluorescence quenching by cocaine binding. Shown are emission spectra of 5 nM h2E2 antibody both before and after addition of 100 nM cocaine, with excitation at either 280 nm (tyrosine and tryptophan) or 295 nm (tryptophan). Note the decrease in emission (quenching) caused by cocaine, but little if any change in the emission maximum (near 330 nm in both cases).

    Techniques Used: Fluorescence, Binding Assay

    10% SDS-PAGE analysis of monoclonal antibody h2E2 with or without treatment with peptide N-glycosidase-F (PNGase-F) to remove all N-linked glycans. After overnight treatment with PNGase-F, either 5 (lanes 2-4), 10 (lanes 5-7), or 20 μg (lanes 8-10) of h2E2 antibody were reduced and denatured in SDS and loaded onto a 10% acrylamide gel, followed by staining with Coomassie Blue R-250. Control, untreated h2E2 (“Con”) along with PNGase-F (“+”) or sham treated (“−”) monoclonal antibody are shown, with the dashed line added to aid visualization of the small differences in electrophoretic mobility observed after N-glycan removal by PNGase-F. The migration positions of the heavy chain, light chain, and PNGase-F enzyme are indicated on the right hand side.
    Figure Legend Snippet: 10% SDS-PAGE analysis of monoclonal antibody h2E2 with or without treatment with peptide N-glycosidase-F (PNGase-F) to remove all N-linked glycans. After overnight treatment with PNGase-F, either 5 (lanes 2-4), 10 (lanes 5-7), or 20 μg (lanes 8-10) of h2E2 antibody were reduced and denatured in SDS and loaded onto a 10% acrylamide gel, followed by staining with Coomassie Blue R-250. Control, untreated h2E2 (“Con”) along with PNGase-F (“+”) or sham treated (“−”) monoclonal antibody are shown, with the dashed line added to aid visualization of the small differences in electrophoretic mobility observed after N-glycan removal by PNGase-F. The migration positions of the heavy chain, light chain, and PNGase-F enzyme are indicated on the right hand side.

    Techniques Used: SDS Page, Acrylamide Gel Assay, Staining, Migration

    5% acrylamide non-equilibrium pH gel electrophoresis (NEPHGE) analysis of monoclonal antibody h2E2. NEPHGE was performed as described in Methods, with the gel run for 90 minutes at 200 V. Aliquots of 5, 10 and 20 μg of h2E2 antibody were analyzed, and the region of the gel containing the separated variants of the monoclonal antibody is expanded on the right side of the figure. Note the separation of several groups of charged variants of the antibody by this technique. BioRad IEF gel standards are shown in the left lane of the gel.
    Figure Legend Snippet: 5% acrylamide non-equilibrium pH gel electrophoresis (NEPHGE) analysis of monoclonal antibody h2E2. NEPHGE was performed as described in Methods, with the gel run for 90 minutes at 200 V. Aliquots of 5, 10 and 20 μg of h2E2 antibody were analyzed, and the region of the gel containing the separated variants of the monoclonal antibody is expanded on the right side of the figure. Note the separation of several groups of charged variants of the antibody by this technique. BioRad IEF gel standards are shown in the left lane of the gel.

    Techniques Used: Nucleic Acid Electrophoresis, Electrofocusing

    Titration of 5 nM h2E2 antibody with cocaine, with excitation at both 280 nm and 295 nm. Only data represented by filled symbols were used for fitting to obtain the indicated sigmoidal curves to derive the EC 50 values. Note the y-axis brake in scale to allow visualization of the quality of the fitted curves for both sets of data on a single plot. K D values were calculated from the EC 50 values and the antibody or Fab concentration as described in Methods. For the experiment shown in the figure (using the intact h2E2 mAb and 295 nm excitation), K D = EC 50 – (0.5 × 2 sites/mAb × 5.0 nM mAb) = 9.4 nM – 5.0 nM = 4.4 nM = K D for cocaine binding to the h2E2 monoclonal antibody.
    Figure Legend Snippet: Titration of 5 nM h2E2 antibody with cocaine, with excitation at both 280 nm and 295 nm. Only data represented by filled symbols were used for fitting to obtain the indicated sigmoidal curves to derive the EC 50 values. Note the y-axis brake in scale to allow visualization of the quality of the fitted curves for both sets of data on a single plot. K D values were calculated from the EC 50 values and the antibody or Fab concentration as described in Methods. For the experiment shown in the figure (using the intact h2E2 mAb and 295 nm excitation), K D = EC 50 – (0.5 × 2 sites/mAb × 5.0 nM mAb) = 9.4 nM – 5.0 nM = 4.4 nM = K D for cocaine binding to the h2E2 monoclonal antibody.

    Techniques Used: Titration, Concentration Assay, Binding Assay

    Reducing 10% SDS-PAGE analysis of h2E2 Fab, mAb and the Endo Lys-C digest. 5 μg of Fab, 6 μg of h2E2 mAb and 6 μg of Endo-Lys-C digested mAb were loaded onto lanes 2-4, respectively. Note the purity and lack of high molecular weight impurities in the Fab preparation. Migration positions of the reduced heavy and light chains, as well as the reduced Fab and Fc fragments, are indicated.
    Figure Legend Snippet: Reducing 10% SDS-PAGE analysis of h2E2 Fab, mAb and the Endo Lys-C digest. 5 μg of Fab, 6 μg of h2E2 mAb and 6 μg of Endo-Lys-C digested mAb were loaded onto lanes 2-4, respectively. Note the purity and lack of high molecular weight impurities in the Fab preparation. Migration positions of the reduced heavy and light chains, as well as the reduced Fab and Fc fragments, are indicated.

    Techniques Used: SDS Page, Molecular Weight, Migration

    8) Product Images from "Necroptotic signaling is primed in Mycobacterium tuberculosis-infected macrophages, but its pathophysiological consequence in disease is restricted"

    Article Title: Necroptotic signaling is primed in Mycobacterium tuberculosis-infected macrophages, but its pathophysiological consequence in disease is restricted

    Journal: Cell Death and Differentiation

    doi: 10.1038/s41418-017-0031-1

    The adaptive immunological response to Mtb infection in vivo is not perturbed by the loss of MLKL signaling a Flow cytometric analysis of the total number of CD4 + T cells, CD8 + T cells and CD19 + MHCII + B cells isolated from the lungs of mice four weeks after infection. b Immunofluorescence staining (blue DAPI, green CD3 and red CD4) of lung sections from mice four weeks after infection. Representative of n = 4 in each group. Three inflammatory lesions were examined per mouse. Scale bar represents 100 μm. c Quantitation of the number of Mtb ESAT-6-specific CD4 + T cells in the lungs of mice four weeks after infection. Cells were isolated from the lungs and restimulated ex vivo with either ESAT-6 or OVA peptide, and analyzed for TNF and IFNγ production by intracellular cytokine staining and flow cytometry. (left) Representative flow cytometry plots and (middle) quantitation of the total number of TNF + IFNγ + CD4 + T cells and (right) their frequency expressed as a percentage of total CD4 + T cells are shown. a , c Graphs show mean and SEM of pooled data from two independent experiments ( n = 6 per group). There were no statistically significant differences between genotypes ( p > 0.05; t test)
    Figure Legend Snippet: The adaptive immunological response to Mtb infection in vivo is not perturbed by the loss of MLKL signaling a Flow cytometric analysis of the total number of CD4 + T cells, CD8 + T cells and CD19 + MHCII + B cells isolated from the lungs of mice four weeks after infection. b Immunofluorescence staining (blue DAPI, green CD3 and red CD4) of lung sections from mice four weeks after infection. Representative of n = 4 in each group. Three inflammatory lesions were examined per mouse. Scale bar represents 100 μm. c Quantitation of the number of Mtb ESAT-6-specific CD4 + T cells in the lungs of mice four weeks after infection. Cells were isolated from the lungs and restimulated ex vivo with either ESAT-6 or OVA peptide, and analyzed for TNF and IFNγ production by intracellular cytokine staining and flow cytometry. (left) Representative flow cytometry plots and (middle) quantitation of the total number of TNF + IFNγ + CD4 + T cells and (right) their frequency expressed as a percentage of total CD4 + T cells are shown. a , c Graphs show mean and SEM of pooled data from two independent experiments ( n = 6 per group). There were no statistically significant differences between genotypes ( p > 0.05; t test)

    Techniques Used: Infection, In Vivo, Flow Cytometry, Isolation, Mouse Assay, Immunofluorescence, Staining, Quantitation Assay, Ex Vivo, Cytometry

    Necroptotic death of Mtb-infected macrophages is restricted a Western blot analysis of markers of programmed cell death in wild-type BMDMs infected with Mtb for 24 h or 48 h. Controls were prepared by treating uninfected BMDMs with either TNF (100 ng/ml), TNF and birinapant (10 μM), or TNF, birinapant and Q-VD-OPh (40 μM) for 24 h. Numbers to the right of the panels represent the positions of protein size markers (in kDa). Representative of three independent experiments. Abbreviations are as follows: T, TNF; S, SMAC mimetic (birinapant); Q, Q-VD-OPh. b Western blot analysis of phosphorylated MLKL in alveolar macrophages isolated from the BAL of uninfected and Mtb-infected wild-type mice. Controls were prepared as in ( a ). Lysates from three uninfected and three infected mice were pooled, and data are representative of two experiments. Vertical dashed lines indicate the positions where lanes that were not contiguous on the blot were juxtaposed to remove unused lanes. c , d Death of wild-type and Mlkl −/ − BMDMs infected with Mtb for 48 h. BMDMs were either ( c) untreated or ( d) treated with 50 ng/ml TNF at 24 h post-infection. The amount of cell death was determined by PI staining and flow cytometry. Graphs show mean and SEM, and data were pooled from three biologically independent experiments. There were no statistically significant differences between genotypes in infected groups ( p > 0.05; t test). e , f Death of primary human monocytes treated with either vehicle (DMSO) or Nec-1s (70 μM) and infected with Mtb for 24 h. Additionally, cells were either ( e) untreated or ( f) treated with 50 ng/ml TNF at 6 h post-infection. The amount of cell death was determined by MTS assay. Graphs show mean and SEM of triplicate wells. Representative of three biologically independent experiments. There were no statistically significant differences between vehicle and Nec-1s-treated cells ( p > 0.05; t test)
    Figure Legend Snippet: Necroptotic death of Mtb-infected macrophages is restricted a Western blot analysis of markers of programmed cell death in wild-type BMDMs infected with Mtb for 24 h or 48 h. Controls were prepared by treating uninfected BMDMs with either TNF (100 ng/ml), TNF and birinapant (10 μM), or TNF, birinapant and Q-VD-OPh (40 μM) for 24 h. Numbers to the right of the panels represent the positions of protein size markers (in kDa). Representative of three independent experiments. Abbreviations are as follows: T, TNF; S, SMAC mimetic (birinapant); Q, Q-VD-OPh. b Western blot analysis of phosphorylated MLKL in alveolar macrophages isolated from the BAL of uninfected and Mtb-infected wild-type mice. Controls were prepared as in ( a ). Lysates from three uninfected and three infected mice were pooled, and data are representative of two experiments. Vertical dashed lines indicate the positions where lanes that were not contiguous on the blot were juxtaposed to remove unused lanes. c , d Death of wild-type and Mlkl −/ − BMDMs infected with Mtb for 48 h. BMDMs were either ( c) untreated or ( d) treated with 50 ng/ml TNF at 24 h post-infection. The amount of cell death was determined by PI staining and flow cytometry. Graphs show mean and SEM, and data were pooled from three biologically independent experiments. There were no statistically significant differences between genotypes in infected groups ( p > 0.05; t test). e , f Death of primary human monocytes treated with either vehicle (DMSO) or Nec-1s (70 μM) and infected with Mtb for 24 h. Additionally, cells were either ( e) untreated or ( f) treated with 50 ng/ml TNF at 6 h post-infection. The amount of cell death was determined by MTS assay. Graphs show mean and SEM of triplicate wells. Representative of three biologically independent experiments. There were no statistically significant differences between vehicle and Nec-1s-treated cells ( p > 0.05; t test)

    Techniques Used: Infection, Western Blot, Isolation, Mouse Assay, Staining, Flow Cytometry, Cytometry, MTS Assay

    9) Product Images from "Proprotein Convertase Processing Enhances Peroxidasin Activity to Reinforce Collagen IV *"

    Article Title: Proprotein Convertase Processing Enhances Peroxidasin Activity to Reinforce Collagen IV *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M116.745935

    Proprotein convertase processing of peroxidasin enhances the formation of sulfilimine cross-links in collagen IV. A and B , CRISPR generated Pxdn knock out PFHR-9 cells ( Control ) were rescued by stable transfection of either wild type ( WT ) or the mutant hPxdn-RGAA ( RGAA ). The matrix containing collagen IV network was isolated 5 days post confluency. The number of cross-links per collagen IV NC1 hexamer was determined using densitometric quantitation of the dimeric and monomeric subunits from SDS-PAGE of the collagenase-digested matrix. C and D , untransfected HEK293 cells or HEK 293 cells stably transfected with either wild type (WT) or the mutant hPxdn-RGAA ( RGAA ) were plated on PFHR-9 uncross-linked matrix, and the underlying matrix was isolated 24 h after overlay for analysis of collagen IV sulfilimine cross-link content. The number of cross-links per collagen IV NC1 hexamer was determined using densitometric quantitation of the dimeric and monomeric subunits from SDS-PAGE of the collagenase-digested matrix. In all panels individual data points are displayed with mean ( dotted line ) and S.D. Data were analyzed using analysis of variance followed by post hoc pairwise comparisons with Bonferroni's correction for multiple comparisons (*, p ≤ 0.05; **, p ≤ 0.01 compared with WT Pxdn).
    Figure Legend Snippet: Proprotein convertase processing of peroxidasin enhances the formation of sulfilimine cross-links in collagen IV. A and B , CRISPR generated Pxdn knock out PFHR-9 cells ( Control ) were rescued by stable transfection of either wild type ( WT ) or the mutant hPxdn-RGAA ( RGAA ). The matrix containing collagen IV network was isolated 5 days post confluency. The number of cross-links per collagen IV NC1 hexamer was determined using densitometric quantitation of the dimeric and monomeric subunits from SDS-PAGE of the collagenase-digested matrix. C and D , untransfected HEK293 cells or HEK 293 cells stably transfected with either wild type (WT) or the mutant hPxdn-RGAA ( RGAA ) were plated on PFHR-9 uncross-linked matrix, and the underlying matrix was isolated 24 h after overlay for analysis of collagen IV sulfilimine cross-link content. The number of cross-links per collagen IV NC1 hexamer was determined using densitometric quantitation of the dimeric and monomeric subunits from SDS-PAGE of the collagenase-digested matrix. In all panels individual data points are displayed with mean ( dotted line ) and S.D. Data were analyzed using analysis of variance followed by post hoc pairwise comparisons with Bonferroni's correction for multiple comparisons (*, p ≤ 0.05; **, p ≤ 0.01 compared with WT Pxdn).

    Techniques Used: CRISPR, Generated, Knock-Out, Stable Transfection, Mutagenesis, Isolation, Quantitation Assay, SDS Page, Transfection

    10) Product Images from "The Yaa locus and IFN? fine tune germinal center B cell selection in murine SLE"

    Article Title: The Yaa locus and IFN? fine tune germinal center B cell selection in murine SLE

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1200745

    Analysis of the Jκ regions associated with (A) Vκ5-43*01 and (B) Vκ5-48*01 light chains in germinal center B cells and hybridomas of male and female 3H9 mice. The amino acid present at the V-J junction (position 116) for each Jκ
    Figure Legend Snippet: Analysis of the Jκ regions associated with (A) Vκ5-43*01 and (B) Vκ5-48*01 light chains in germinal center B cells and hybridomas of male and female 3H9 mice. The amino acid present at the V-J junction (position 116) for each Jκ

    Techniques Used: Mouse Assay

    Repertoire analysis of 3H9-associated Vκ chains in aged male (A) and female (B) NZW/BXSB mice. Genes that contribute > 5% of the χ 2 value and constitute > 2.5% of the repertoire in any of the comparisons are shown. 39–93
    Figure Legend Snippet: Repertoire analysis of 3H9-associated Vκ chains in aged male (A) and female (B) NZW/BXSB mice. Genes that contribute > 5% of the χ 2 value and constitute > 2.5% of the repertoire in any of the comparisons are shown. 39–93

    Techniques Used: Mouse Assay

    11) Product Images from "Cooperation among Multiple Transcription Factors Is Required for Access to Minimal T-Cell Receptor ?-Enhancer Chromatin In Vivo"

    Article Title: Cooperation among Multiple Transcription Factors Is Required for Access to Minimal T-Cell Receptor ?-Enhancer Chromatin In Vivo

    Journal: Molecular and Cellular Biology

    doi:

    Local chromatin disruption by the wild-type minimal Eα. Transgenic thymocyte DNAs from wild-type Tα1,2 line T2 and Tα1,2mTCF/LEF line JJ were digested with DNase I either as naked DNA in vitro or as chromatin in permeabilized cells. DNA samples (10 μg) were digested with Sac I, electrophoresed through a 0.9% agarose gel, and analyzed on a Southern blot probed with a 32 P-labeled 1.1-kb J δ ). A DNase I-hypersensitive region over the enhancer in line T2 is denoted by a bracket. Size makers (in kilobases) are indicated at the left.
    Figure Legend Snippet: Local chromatin disruption by the wild-type minimal Eα. Transgenic thymocyte DNAs from wild-type Tα1,2 line T2 and Tα1,2mTCF/LEF line JJ were digested with DNase I either as naked DNA in vitro or as chromatin in permeabilized cells. DNA samples (10 μg) were digested with Sac I, electrophoresed through a 0.9% agarose gel, and analyzed on a Southern blot probed with a 32 P-labeled 1.1-kb J δ ). A DNase I-hypersensitive region over the enhancer in line T2 is denoted by a bracket. Size makers (in kilobases) are indicated at the left.

    Techniques Used: Transgenic Assay, In Vitro, Agarose Gel Electrophoresis, Southern Blot, Labeling

    12) Product Images from "Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells"

    Article Title: Differential Nucleosome Occupancies across Oct4-Sox2 Binding Sites in Murine Embryonic Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0127214

    Mapping in vivo and in vitro nucleosome occupancy using BAC-based enrichment. (a) Protocols were modified so that permeabilization and micrococcal nuclease digestion occur while embryonic stem cells are attached to the tissue culture surface to improve recovery and digestion reproducibility. The amount of micrococcal nuclease (MNase) in the digestion was titrated so that the mononucleosome band at 147bp is the primary band, without overdigestion. Digests were measured in Worthington Units of MNase * Time of digestion / volume of cell culture (U*min/mL). Lanes 1 and 8: 50bp ladder. Lanes 2–7 range from 2500 U*min/mL to 25,000 U*min/mL of MNase, with ideal digestion in the third condition, 10,000 U*min/mL. This amount of digestion was used in all future experiments. (b) Genomic DNA was purified from embryonic stem cell cultures and combined with histone octamer purified from chicken erythrocytes in a ratio of 100μg:30μg under high salt (2M NaCl) conditions. Removal of salt via dialysis results in reconstituted chromatin, representing histone proteins’ preferred DNA sequences. Reconstituted chromatin was digested with 5 Worthington Units of micrococcal nuclease per 10μg of genomic DNA present, for a digestion of 5 minutes at 37°C (Lanes 1 and 7: 50 bp ladder; Lanes 2–6: digested chromatin). (c) Bacterial Artificial Chromosome (BAC) DNA, which was nicked with biotin-dUTP, was blocked with Cot-1 DNA at a ratio of 100ng:10μg. 1μg of library-adapted mononucleosome DNA was denatured and mixed with BAC DNA. Mononucleosome DNA was hybridized to the corresponding BAC region and was isolated by removing BACs from solution with streptavadin beads, stringently washing the beads, and eluting single stranded DNA from the beads. Double stranded products were amplified using PCR and sent for paired-end sequencing.
    Figure Legend Snippet: Mapping in vivo and in vitro nucleosome occupancy using BAC-based enrichment. (a) Protocols were modified so that permeabilization and micrococcal nuclease digestion occur while embryonic stem cells are attached to the tissue culture surface to improve recovery and digestion reproducibility. The amount of micrococcal nuclease (MNase) in the digestion was titrated so that the mononucleosome band at 147bp is the primary band, without overdigestion. Digests were measured in Worthington Units of MNase * Time of digestion / volume of cell culture (U*min/mL). Lanes 1 and 8: 50bp ladder. Lanes 2–7 range from 2500 U*min/mL to 25,000 U*min/mL of MNase, with ideal digestion in the third condition, 10,000 U*min/mL. This amount of digestion was used in all future experiments. (b) Genomic DNA was purified from embryonic stem cell cultures and combined with histone octamer purified from chicken erythrocytes in a ratio of 100μg:30μg under high salt (2M NaCl) conditions. Removal of salt via dialysis results in reconstituted chromatin, representing histone proteins’ preferred DNA sequences. Reconstituted chromatin was digested with 5 Worthington Units of micrococcal nuclease per 10μg of genomic DNA present, for a digestion of 5 minutes at 37°C (Lanes 1 and 7: 50 bp ladder; Lanes 2–6: digested chromatin). (c) Bacterial Artificial Chromosome (BAC) DNA, which was nicked with biotin-dUTP, was blocked with Cot-1 DNA at a ratio of 100ng:10μg. 1μg of library-adapted mononucleosome DNA was denatured and mixed with BAC DNA. Mononucleosome DNA was hybridized to the corresponding BAC region and was isolated by removing BACs from solution with streptavadin beads, stringently washing the beads, and eluting single stranded DNA from the beads. Double stranded products were amplified using PCR and sent for paired-end sequencing.

    Techniques Used: In Vivo, In Vitro, BAC Assay, Modification, Cell Culture, Purification, Isolation, Amplification, Polymerase Chain Reaction, Sequencing

    13) Product Images from "The Ancient Immunoglobulin Domains of Peroxidasin Are Required to Form Sulfilimine Cross-links in Collagen IV *"

    Article Title: The Ancient Immunoglobulin Domains of Peroxidasin Are Required to Form Sulfilimine Cross-links in Collagen IV *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.673996

    Peroxidasin uniquely forms sulfilimine cross-links in collagen IV. a, experimental design of overlay experiments. PFHR-9 cells were grown in the presence of 50 μ m phloroglucinol (peroxidase inhibitor) to deposit an uncross-linked collagen IV network.
    Figure Legend Snippet: Peroxidasin uniquely forms sulfilimine cross-links in collagen IV. a, experimental design of overlay experiments. PFHR-9 cells were grown in the presence of 50 μ m phloroglucinol (peroxidase inhibitor) to deposit an uncross-linked collagen IV network.

    Techniques Used:

    The immunoglobulin and peroxidase domains of peroxidasin are required for sulfilimine bond formation. a, schematic depiction of the domain structure of peroxidasin and its deletion constructs. b, immunoblotting of media and matrix fractions under reducing
    Figure Legend Snippet: The immunoglobulin and peroxidase domains of peroxidasin are required for sulfilimine bond formation. a, schematic depiction of the domain structure of peroxidasin and its deletion constructs. b, immunoblotting of media and matrix fractions under reducing

    Techniques Used: Construct

    Evolution of animal peroxidases and model for peroxidasin-mediated sulfilimine bond formation in collagen IV. a, PXDN is the ancestral animal heme peroxidase found in Cnidaria that extends into invertebrates and vertebrates with additional Ig and vWFC
    Figure Legend Snippet: Evolution of animal peroxidases and model for peroxidasin-mediated sulfilimine bond formation in collagen IV. a, PXDN is the ancestral animal heme peroxidase found in Cnidaria that extends into invertebrates and vertebrates with additional Ig and vWFC

    Techniques Used:

    14) Product Images from "The spring-loaded genome: Nucleosome redistributions are widespread, transient, and DNA-directed"

    Article Title: The spring-loaded genome: Nucleosome redistributions are widespread, transient, and DNA-directed

    Journal: Genome Research

    doi: 10.1101/gr.160150.113

    Nucleosome redistributions are determined by the underlying DNA sequence. ( A ) Western blots with the specified antibodies, at various times (in hours) after KSHV reactivation (hpr), of iSLK.219 cells treated with 0.2 µg/mL doxycycline. BRG1 protein
    Figure Legend Snippet: Nucleosome redistributions are determined by the underlying DNA sequence. ( A ) Western blots with the specified antibodies, at various times (in hours) after KSHV reactivation (hpr), of iSLK.219 cells treated with 0.2 µg/mL doxycycline. BRG1 protein

    Techniques Used: Sequencing, Western Blot

    15) Product Images from "The Immunoglobulin Heavy Chain Locus Control Region Increases Histone Acetylation along Linked c-myc Genes"

    Article Title: The Immunoglobulin Heavy Chain Locus Control Region Increases Histone Acetylation along Linked c-myc Genes

    Journal: Molecular and Cellular Biology

    doi:

    The HS1234 enhancer has little effect on the chromatin organization of linked c- myc genes on episomal templates. (A) DNase I mapping of HSs in the upstream and promoter regions of control and enhancer-linked templates. Genomic DNAs purified from DNase I-treated nuclei were digested with Bgl 2 and used in a Southern analysis with probe a, a Bgl 2- Eco RV fragment indicated on the map shown in panel B. Major HSs I, II 2 , III 1 , III 2 , and V are indicated, and minor sites within the first intron are denoted by open circles. Hybridization fragments larger than 5,385 bp originate from vector or HS1234 sequences upstream of the c- myc Hin dIII site. (B) Map of restriction sites and probes used in these analyses. (C and D) MNase mapping of nucleosome positioning along control and HS1234-linked c- myc episomes. Genomic DNAs purified from MNase-treated nuclei were digested with either Sty1 (C) or Xba I (D) and used in Southern analyses with probes b and c, as indicated below the blots and on the map in panel B. The locations of upstream regulatory sequences, the P1 and P2 promoters, and the exon 1-intron 1 junction (Int1/Ex1) are indicated alongside the blots. Region of increased MNase sensitivity along HS1234-linked templates within the c- myc promoter and downstream of the transcriptional start sites is indicated by the asterisk in panel D. WT, wild type.
    Figure Legend Snippet: The HS1234 enhancer has little effect on the chromatin organization of linked c- myc genes on episomal templates. (A) DNase I mapping of HSs in the upstream and promoter regions of control and enhancer-linked templates. Genomic DNAs purified from DNase I-treated nuclei were digested with Bgl 2 and used in a Southern analysis with probe a, a Bgl 2- Eco RV fragment indicated on the map shown in panel B. Major HSs I, II 2 , III 1 , III 2 , and V are indicated, and minor sites within the first intron are denoted by open circles. Hybridization fragments larger than 5,385 bp originate from vector or HS1234 sequences upstream of the c- myc Hin dIII site. (B) Map of restriction sites and probes used in these analyses. (C and D) MNase mapping of nucleosome positioning along control and HS1234-linked c- myc episomes. Genomic DNAs purified from MNase-treated nuclei were digested with either Sty1 (C) or Xba I (D) and used in Southern analyses with probes b and c, as indicated below the blots and on the map in panel B. The locations of upstream regulatory sequences, the P1 and P2 promoters, and the exon 1-intron 1 junction (Int1/Ex1) are indicated alongside the blots. Region of increased MNase sensitivity along HS1234-linked templates within the c- myc promoter and downstream of the transcriptional start sites is indicated by the asterisk in panel D. WT, wild type.

    Techniques Used: Purification, Hybridization, Plasmid Preparation

    16) Product Images from "Histone modifications silence the GATA transcription factor genes in ovarian cancer"

    Article Title: Histone modifications silence the GATA transcription factor genes in ovarian cancer

    Journal: Oncogene

    doi: 10.1038/sj.onc.1209533

    DNase I hypersensitivity of GATA4 and GATA6 promoters in ovarian cancer cells and effect of trichostatin A (TSA). (a) Schematic illustration of the GATA4 promoter region indicating the restriction sites of the Xmn I (X) restriction enzyme used for complete digestion and location of the probe (1260bp) used for Southern blot hybridization. (b) DNase I hypersensitivity assay of the GATA4 promoter in ES2 and A2780 cells cultured without or with TSA. The parental band indicated (arrow) is the Xmn I fragment including exon 1 of the GATA4 gene. (c) Schematic illustration of the GATA6 promoter region indicating the restriction sites of the Bam H1 (b) , and location of the probe (985bp) used for Southern blot hybridization. (d) DNase I hypersensitivity assay of the GATA6 promoter in ES2 and A2780 cells treated without or with TSA. Parental band (arrow) and DNase I hypersensitivity bands (*) are indicated. The experiment was repeated and a similar conclusion was reached.
    Figure Legend Snippet: DNase I hypersensitivity of GATA4 and GATA6 promoters in ovarian cancer cells and effect of trichostatin A (TSA). (a) Schematic illustration of the GATA4 promoter region indicating the restriction sites of the Xmn I (X) restriction enzyme used for complete digestion and location of the probe (1260bp) used for Southern blot hybridization. (b) DNase I hypersensitivity assay of the GATA4 promoter in ES2 and A2780 cells cultured without or with TSA. The parental band indicated (arrow) is the Xmn I fragment including exon 1 of the GATA4 gene. (c) Schematic illustration of the GATA6 promoter region indicating the restriction sites of the Bam H1 (b) , and location of the probe (985bp) used for Southern blot hybridization. (d) DNase I hypersensitivity assay of the GATA6 promoter in ES2 and A2780 cells treated without or with TSA. Parental band (arrow) and DNase I hypersensitivity bands (*) are indicated. The experiment was repeated and a similar conclusion was reached.

    Techniques Used: Southern Blot, Hybridization, Cell Culture

    17) Product Images from "Preparation of Soluble Proteins from Escherichia coli"

    Article Title: Preparation of Soluble Proteins from Escherichia coli

    Journal: Current protocols in protein science / editorial board, John E. Coligan ... [et al.]

    doi: 10.1002/0471140864.ps0602s78

    Chromatofocusing of IL-1β. ( A ) Theoretical titration curve of IL-1β with N-terminal methionine (M + ) and N-terminal alanine (M − ). ( B ) Chromatofocusing of a mixture of IL-1β M + and IL-1β M − . Chromatography
    Figure Legend Snippet: Chromatofocusing of IL-1β. ( A ) Theoretical titration curve of IL-1β with N-terminal methionine (M + ) and N-terminal alanine (M − ). ( B ) Chromatofocusing of a mixture of IL-1β M + and IL-1β M − . Chromatography

    Techniques Used: Titration, Chromatography

    Scheme for purifying human interleukin-1β.
    Figure Legend Snippet: Scheme for purifying human interleukin-1β.

    Techniques Used:

    Purification of IL-1β. ( A ) SDS-PAGE analysis of samples at various stages. Analysis was conducted on a gel of dimensions 12 cm × 16 cm × 1.5 mm. Lane a, purified protein (100 μg loaded); lane b, purified protein (10 μg
    Figure Legend Snippet: Purification of IL-1β. ( A ) SDS-PAGE analysis of samples at various stages. Analysis was conducted on a gel of dimensions 12 cm × 16 cm × 1.5 mm. Lane a, purified protein (100 μg loaded); lane b, purified protein (10 μg

    Techniques Used: Purification, SDS Page

    18) Product Images from "Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment"

    Article Title: Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment

    Journal: Human Vaccines & Immunotherapeutics

    doi: 10.4161/21645515.2014.990856

    High performance strong cation exchange chromatography of the h2E2 antibody before and after treatment with carboxypeptidase B. 100 μg of h2E2 antibody was injected, and eluted with a gradient of NaCl in MES buffer. Note the disappearance of peaks 1 and 2 after removal of the C-terminal lysine residues. These peaks represent h2E2 antibody molecules containing 1 or 2 lysine residues on the C-termini of the 2 heavy chains in the antibody (which, after removal of lysine by carboxypeptidase elute with the main peak labeled ‘0” eluting at approximately 11 minutes).
    Figure Legend Snippet: High performance strong cation exchange chromatography of the h2E2 antibody before and after treatment with carboxypeptidase B. 100 μg of h2E2 antibody was injected, and eluted with a gradient of NaCl in MES buffer. Note the disappearance of peaks 1 and 2 after removal of the C-terminal lysine residues. These peaks represent h2E2 antibody molecules containing 1 or 2 lysine residues on the C-termini of the 2 heavy chains in the antibody (which, after removal of lysine by carboxypeptidase elute with the main peak labeled ‘0” eluting at approximately 11 minutes).

    Techniques Used: Chromatography, Injection, Labeling

    19) Product Images from "An intersubunit contact stimulating transcription initiation by E. coli RNA polymerase: interaction of the ? C-terminal domain and ? region 4"

    Article Title: An intersubunit contact stimulating transcription initiation by E. coli RNA polymerase: interaction of the ? C-terminal domain and ? region 4

    Journal: Genes & Development

    doi: 10.1101/gad.1079403

    DNAse I footprints of promoters in the presence of wild-type and mutant RNAPs. Footprints were performed on rrnB P1 containing UP element 4547 ( A ), UP element 4549 ( B ), the rrnB P1 full UP element ( C ), or the lacUV5 promoter ( D ; see Materials and Methods). The regions in DNA protected from DNAse I cleavage and the regions corresponding to the UP elements are indicated under the gel images. The RNAP used in each lane is indicated at the left of the gel images: wild-type RNAP (WT), αE261A RNAP (αE261A), σ R603A RNAP (σR603A), or no RNAP (−). Superimposed scans of gel lanes are shown above the images. Scans corresponding to footprints with mutant RNAPs are red, footprints with wild-type RNAP are blue, and footprints without RNAP are gray. Red arrows indicate position −38, and this region is magnified in the inset in each panel. Footprints of an E261A RNAP–4549 promoter complex were very similar to those shown in A , and footprints on an E261A RNAP– rrnB P1 full UP element promoter complex were very similar to those shown in C (data not shown). On the 4547 ( A ) and 4549 ( B ) promoters, position −38 was more sensitive to DNAse I cleavage in the complexes formed with the mutant RNAPs than with the wild-type RNAP.
    Figure Legend Snippet: DNAse I footprints of promoters in the presence of wild-type and mutant RNAPs. Footprints were performed on rrnB P1 containing UP element 4547 ( A ), UP element 4549 ( B ), the rrnB P1 full UP element ( C ), or the lacUV5 promoter ( D ; see Materials and Methods). The regions in DNA protected from DNAse I cleavage and the regions corresponding to the UP elements are indicated under the gel images. The RNAP used in each lane is indicated at the left of the gel images: wild-type RNAP (WT), αE261A RNAP (αE261A), σ R603A RNAP (σR603A), or no RNAP (−). Superimposed scans of gel lanes are shown above the images. Scans corresponding to footprints with mutant RNAPs are red, footprints with wild-type RNAP are blue, and footprints without RNAP are gray. Red arrows indicate position −38, and this region is magnified in the inset in each panel. Footprints of an E261A RNAP–4549 promoter complex were very similar to those shown in A , and footprints on an E261A RNAP– rrnB P1 full UP element promoter complex were very similar to those shown in C (data not shown). On the 4547 ( A ) and 4549 ( B ) promoters, position −38 was more sensitive to DNAse I cleavage in the complexes formed with the mutant RNAPs than with the wild-type RNAP.

    Techniques Used: Mutagenesis

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