Structured Review

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Protease accessibility of newly synthesized Cytb5 is altered by Ca 2+ -CaM. Cytb5 was synthesized in the presence of [ 35 S]methionine and divided into three aliquots. The three translation reactions were supplemented with buffer, Ca 2+ -CaM, and TFP as indicated and incubated for 30 min at 30 °C. Subsequently, each of the three translation reactions was divided into five aliquots and incubated with increasing dilutions of proteinase K for 60 min at 0 °C as indicated (starting concentration 175 μg/ml). After protease inhibition, all samples were subjected to SDS–PAGE and <t>phosphorimaging.</t> Only the areas of interest of a single gel are shown. The experiment was carried out three times with similar results.
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Images

1) Product Images from "Ca2+-calmodulin inhibits tail-anchored protein insertion into the mammalian endoplasmic reticulum membrane"

Article Title: Ca2+-calmodulin inhibits tail-anchored protein insertion into the mammalian endoplasmic reticulum membrane

Journal: Febs Letters

doi: 10.1016/j.febslet.2011.10.008

Protease accessibility of newly synthesized Cytb5 is altered by Ca 2+ -CaM. Cytb5 was synthesized in the presence of [ 35 S]methionine and divided into three aliquots. The three translation reactions were supplemented with buffer, Ca 2+ -CaM, and TFP as indicated and incubated for 30 min at 30 °C. Subsequently, each of the three translation reactions was divided into five aliquots and incubated with increasing dilutions of proteinase K for 60 min at 0 °C as indicated (starting concentration 175 μg/ml). After protease inhibition, all samples were subjected to SDS–PAGE and phosphorimaging. Only the areas of interest of a single gel are shown. The experiment was carried out three times with similar results.
Figure Legend Snippet: Protease accessibility of newly synthesized Cytb5 is altered by Ca 2+ -CaM. Cytb5 was synthesized in the presence of [ 35 S]methionine and divided into three aliquots. The three translation reactions were supplemented with buffer, Ca 2+ -CaM, and TFP as indicated and incubated for 30 min at 30 °C. Subsequently, each of the three translation reactions was divided into five aliquots and incubated with increasing dilutions of proteinase K for 60 min at 0 °C as indicated (starting concentration 175 μg/ml). After protease inhibition, all samples were subjected to SDS–PAGE and phosphorimaging. Only the areas of interest of a single gel are shown. The experiment was carried out three times with similar results.

Techniques Used: Synthesized, Chick Chorioallantoic Membrane Assay, Incubation, Concentration Assay, Inhibition, SDS Page

Binding of Syb2 to TRC40/Asna-1 is displaced by Ca 2+ -CaM. Syb2 was synthesized in the presence of [ 35 S]methionine. The translation reaction was divided into aliquots, then supplemented and incubated with buffer, Ca 2+ -CaM, Ca 2+ -GST-CaM, GST, TFP, and apyrase as indicated. The final concentrations of GST and GST-CaM were 0.26 mg/ml. The aliquots were then incubated with the heterobifunctional cross-linking reagent EDC and all samples were subjected to SDS–PAGE and phosphorimaging. The positions of molecular mass standards are indicated (kDa). The experiment was carried out three times with similar results. We note that GST-CaM was equally efficient in inhibiting membrane insertion of TA proteins in the presence of Ca 2+ as compared to CaM and that this inhibitory effect was suppressed by TFP (data not shown).
Figure Legend Snippet: Binding of Syb2 to TRC40/Asna-1 is displaced by Ca 2+ -CaM. Syb2 was synthesized in the presence of [ 35 S]methionine. The translation reaction was divided into aliquots, then supplemented and incubated with buffer, Ca 2+ -CaM, Ca 2+ -GST-CaM, GST, TFP, and apyrase as indicated. The final concentrations of GST and GST-CaM were 0.26 mg/ml. The aliquots were then incubated with the heterobifunctional cross-linking reagent EDC and all samples were subjected to SDS–PAGE and phosphorimaging. The positions of molecular mass standards are indicated (kDa). The experiment was carried out three times with similar results. We note that GST-CaM was equally efficient in inhibiting membrane insertion of TA proteins in the presence of Ca 2+ as compared to CaM and that this inhibitory effect was suppressed by TFP (data not shown).

Techniques Used: Binding Assay, Chick Chorioallantoic Membrane Assay, Synthesized, Incubation, SDS Page

Insertion of model tail-anchored (TA) membrane proteins into rough microsomes is inhibited by Ca 2+ -CaM. TA proteins were synthesized in the presence of [ 35 S]methionine. After inhibition of protein synthesis, the translation reactions were supplemented with buffer (lane 1) and canine pancreatic microsomes (RM) or semi-permeabilized HeLa cells (SPC) (lanes 2–5) and divided into aliquots. Where indicated, the aliquots were supplemented simultaneously with apyrase, Ca 2+ , CaM, and TFP. The experiment that is depicted in d was carried out in the presence of the acceptor tripeptide NYT to prevent N-glycosylation. After further incubation for 30 min, samples were left untreated (a,g) or subjected to either centrifugation (b,c) or carbonate extraction (d–f). Samples or sample pellets after centrifugation or carbonate extraction were subjected to SDS–PAGE and phosphorimaging. Only the areas of interest in single gels are shown in the left panels. The right panels show the mean values and the standard errors of the mean from at least four individual experiments. The amount of carbonate-resistant protein was corrected using the buffer background value. The efficiency of membrane integration for the buffer control (as assayed as either N-glycosylation or carbonate resistance) was set as 100%.
Figure Legend Snippet: Insertion of model tail-anchored (TA) membrane proteins into rough microsomes is inhibited by Ca 2+ -CaM. TA proteins were synthesized in the presence of [ 35 S]methionine. After inhibition of protein synthesis, the translation reactions were supplemented with buffer (lane 1) and canine pancreatic microsomes (RM) or semi-permeabilized HeLa cells (SPC) (lanes 2–5) and divided into aliquots. Where indicated, the aliquots were supplemented simultaneously with apyrase, Ca 2+ , CaM, and TFP. The experiment that is depicted in d was carried out in the presence of the acceptor tripeptide NYT to prevent N-glycosylation. After further incubation for 30 min, samples were left untreated (a,g) or subjected to either centrifugation (b,c) or carbonate extraction (d–f). Samples or sample pellets after centrifugation or carbonate extraction were subjected to SDS–PAGE and phosphorimaging. Only the areas of interest in single gels are shown in the left panels. The right panels show the mean values and the standard errors of the mean from at least four individual experiments. The amount of carbonate-resistant protein was corrected using the buffer background value. The efficiency of membrane integration for the buffer control (as assayed as either N-glycosylation or carbonate resistance) was set as 100%.

Techniques Used: Chick Chorioallantoic Membrane Assay, Synthesized, Inhibition, Incubation, Centrifugation, SDS Page

2) Product Images from "Mammalian SRP receptor switches the Sec61 translocase from Sec62 to SRP-dependent translocation"

Article Title: Mammalian SRP receptor switches the Sec61 translocase from Sec62 to SRP-dependent translocation

Journal: Nature Communications

doi: 10.1038/ncomms10133

SR can inhibit translocation of Sec62-dependent precursors. ( a ) Constructs of apelin, statherin, preprocecropin A (ppcec A) and cytochrome B5 (cyt B5) each with a C-terminal opsin tag containing two N-linked glycosylation sites (OPG2) were translated in vitro in rabbit reticulocyte lysate in the presence of [ 35 S] methionine. Synthesis was terminated with puromycin to ensure release of all nascent chains from the ribosome. PKRM were then added in the presence of absence of SRα/βΔN (10 μM) and then incubated at 30 °C to permit targeting and translocation. Membranes were then reisolated through a sucrose cushion and analysed by SDS–PAGE and phosphorimaging. The position of unglycosylated non signal-sequence cleaved (*) and signal-sequence cleaved, twice glycosylated species ( > ) is indicated. ( b ) Preprocecropin A (ppCec A) and cytochrome B5 both with a C-terminal opsin tag (OPG2) as well as preprolactin (pPL) were translated in reticulocyte lysate in the presense [ 35 S] methionine and microsomes that had been preincubated with either buffer or the different SR constructs. Processed and non-processed forms of each precursor were recovered by denaturing immuno-precipitation and analysed by SDS–PAGE and phosphorimaging. ( c ) Relative translocation efficiency was determined from the ratio of processed to non-processed form for each precursor (as in b ). Translocation in the absence of recombinant SR was set to 100%. Data are the means of three independent experiments. Error bars represent s.e.m. Differences significant from the buffer control are indicated (one-way analysis of variance, * P
Figure Legend Snippet: SR can inhibit translocation of Sec62-dependent precursors. ( a ) Constructs of apelin, statherin, preprocecropin A (ppcec A) and cytochrome B5 (cyt B5) each with a C-terminal opsin tag containing two N-linked glycosylation sites (OPG2) were translated in vitro in rabbit reticulocyte lysate in the presence of [ 35 S] methionine. Synthesis was terminated with puromycin to ensure release of all nascent chains from the ribosome. PKRM were then added in the presence of absence of SRα/βΔN (10 μM) and then incubated at 30 °C to permit targeting and translocation. Membranes were then reisolated through a sucrose cushion and analysed by SDS–PAGE and phosphorimaging. The position of unglycosylated non signal-sequence cleaved (*) and signal-sequence cleaved, twice glycosylated species ( > ) is indicated. ( b ) Preprocecropin A (ppCec A) and cytochrome B5 both with a C-terminal opsin tag (OPG2) as well as preprolactin (pPL) were translated in reticulocyte lysate in the presense [ 35 S] methionine and microsomes that had been preincubated with either buffer or the different SR constructs. Processed and non-processed forms of each precursor were recovered by denaturing immuno-precipitation and analysed by SDS–PAGE and phosphorimaging. ( c ) Relative translocation efficiency was determined from the ratio of processed to non-processed form for each precursor (as in b ). Translocation in the absence of recombinant SR was set to 100%. Data are the means of three independent experiments. Error bars represent s.e.m. Differences significant from the buffer control are indicated (one-way analysis of variance, * P

Techniques Used: Translocation Assay, Construct, In Vitro, Incubation, SDS Page, Sequencing, Immunoprecipitation, Recombinant

3) Product Images from "Mammalian SRP receptor switches the Sec61 translocase from Sec62 to SRP-dependent translocation"

Article Title: Mammalian SRP receptor switches the Sec61 translocase from Sec62 to SRP-dependent translocation

Journal: Nature Communications

doi: 10.1038/ncomms10133

SR can inhibit translocation of Sec62-dependent precursors. ( a ) Constructs of apelin, statherin, preprocecropin A (ppcec A) and cytochrome B5 (cyt B5) each with a C-terminal opsin tag containing two N-linked glycosylation sites (OPG2) were translated in vitro in rabbit reticulocyte lysate in the presence of [ 35 S] methionine. Synthesis was terminated with puromycin to ensure release of all nascent chains from the ribosome. PKRM were then added in the presence of absence of SRα/βΔN (10 μM) and then incubated at 30 °C to permit targeting and translocation. Membranes were then reisolated through a sucrose cushion and analysed by SDS–PAGE and phosphorimaging. The position of unglycosylated non signal-sequence cleaved (*) and signal-sequence cleaved, twice glycosylated species ( > ) is indicated. ( b ) Preprocecropin A (ppCec A) and cytochrome B5 both with a C-terminal opsin tag (OPG2) as well as preprolactin (pPL) were translated in reticulocyte lysate in the presense [ 35 S] methionine and microsomes that had been preincubated with either buffer or the different SR constructs. Processed and non-processed forms of each precursor were recovered by denaturing immuno-precipitation and analysed by SDS–PAGE and phosphorimaging. ( c ) Relative translocation efficiency was determined from the ratio of processed to non-processed form for each precursor (as in b ). Translocation in the absence of recombinant SR was set to 100%. Data are the means of three independent experiments. Error bars represent s.e.m. Differences significant from the buffer control are indicated (one-way analysis of variance, * P
Figure Legend Snippet: SR can inhibit translocation of Sec62-dependent precursors. ( a ) Constructs of apelin, statherin, preprocecropin A (ppcec A) and cytochrome B5 (cyt B5) each with a C-terminal opsin tag containing two N-linked glycosylation sites (OPG2) were translated in vitro in rabbit reticulocyte lysate in the presence of [ 35 S] methionine. Synthesis was terminated with puromycin to ensure release of all nascent chains from the ribosome. PKRM were then added in the presence of absence of SRα/βΔN (10 μM) and then incubated at 30 °C to permit targeting and translocation. Membranes were then reisolated through a sucrose cushion and analysed by SDS–PAGE and phosphorimaging. The position of unglycosylated non signal-sequence cleaved (*) and signal-sequence cleaved, twice glycosylated species ( > ) is indicated. ( b ) Preprocecropin A (ppCec A) and cytochrome B5 both with a C-terminal opsin tag (OPG2) as well as preprolactin (pPL) were translated in reticulocyte lysate in the presense [ 35 S] methionine and microsomes that had been preincubated with either buffer or the different SR constructs. Processed and non-processed forms of each precursor were recovered by denaturing immuno-precipitation and analysed by SDS–PAGE and phosphorimaging. ( c ) Relative translocation efficiency was determined from the ratio of processed to non-processed form for each precursor (as in b ). Translocation in the absence of recombinant SR was set to 100%. Data are the means of three independent experiments. Error bars represent s.e.m. Differences significant from the buffer control are indicated (one-way analysis of variance, * P

Techniques Used: Translocation Assay, Construct, In Vitro, Incubation, SDS Page, Sequencing, Immunoprecipitation, Recombinant

4) Product Images from "Polyketide Chain Skipping Mechanism in the Biosynthesis of the Hybrid Nonribosomal Peptide-Polyketide Antitumor Antibiotic Leinamycin in Streptomyces atroolivaceus S-140 ⊥"

Article Title: Polyketide Chain Skipping Mechanism in the Biosynthesis of the Hybrid Nonribosomal Peptide-Polyketide Antitumor Antibiotic Leinamycin in Streptomyces atroolivaceus S-140 ⊥

Journal:

doi: 10.1021/np050467t

In vitro assays of LnmG-catalyzed loading of malonyl CoA to LnmJ ACP 6-1 and ACP 6-2 . (A) Incubation of ACP 6-1 and ACP 6-2 with [2- 14 C]malonyl CoA and LnmG as visualized on a 4– 15 % SDSPAGE (I) and by phosphorimaging (II). Lane 1, molecular weight
Figure Legend Snippet: In vitro assays of LnmG-catalyzed loading of malonyl CoA to LnmJ ACP 6-1 and ACP 6-2 . (A) Incubation of ACP 6-1 and ACP 6-2 with [2- 14 C]malonyl CoA and LnmG as visualized on a 4– 15 % SDSPAGE (I) and by phosphorimaging (II). Lane 1, molecular weight

Techniques Used: In Vitro, Incubation, Molecular Weight

5) Product Images from "Structural mimicry in transcription regulation of human RNA polymerase II by the DNA helicase RECQL5"

Article Title: Structural mimicry in transcription regulation of human RNA polymerase II by the DNA helicase RECQL5

Journal: Nature structural & molecular biology

doi: 10.1038/nsmb.2596

Both RECQL5 helicase and IRI domains are required for repression of transcription ( a ) Schematic overview of the in vitro transcription assay. The dC–tailed histone H3.3 intron DNA was used as template and transcribed by purified Pol II (0.8 pmol) in the presence of recombinant wild–type and mutant RECQL5 proteins at two different concentrations (0.2 µM and 2 µM, corresponding to a 5–fold and 50–fold molar excess over Pol II, respectively). ( b ) Transcription assay performed as outlined in (a) in the presence of recombinant RECQL5 fragments and their mutants, as indicated. Transcripts were resolved by electrophoresis on a denaturing polyacrylamide gel and visualized by phosphorimaging. The transcription template contains three stall sites, giving rise to stalled transcripts TIa, TIb, and TII in addition to the run–off transcript (RO).
Figure Legend Snippet: Both RECQL5 helicase and IRI domains are required for repression of transcription ( a ) Schematic overview of the in vitro transcription assay. The dC–tailed histone H3.3 intron DNA was used as template and transcribed by purified Pol II (0.8 pmol) in the presence of recombinant wild–type and mutant RECQL5 proteins at two different concentrations (0.2 µM and 2 µM, corresponding to a 5–fold and 50–fold molar excess over Pol II, respectively). ( b ) Transcription assay performed as outlined in (a) in the presence of recombinant RECQL5 fragments and their mutants, as indicated. Transcripts were resolved by electrophoresis on a denaturing polyacrylamide gel and visualized by phosphorimaging. The transcription template contains three stall sites, giving rise to stalled transcripts TIa, TIb, and TII in addition to the run–off transcript (RO).

Techniques Used: In Vitro, Purification, Recombinant, Mutagenesis, Electrophoresis

RECQL5 IRI domain competes with TFIIS for binding to Pol II and inhibits TFIIS–mediated read–through of intrinsic elongation blocks ( a ) Pull-down assay probing Pol II binding to GST–TFIIS in the presence of either wild–type or K598E mutant RECQL5 IRI domains. Bound proteins were analyzed by SDS–PAGE, followed by staining with Coomassie Blue or Western blotting using an anti–Rbp1 antibody. ( b ) Schematic overview of the pulse–chase TFIIS read–through transcription assay using histone H3.3 intron DNA template. Following addition of chase buffer, RECQL5 variants (0.1 µM, 1.0 µM and 10 µM final concentration, corresponding to 5–, 50– and 500–fold molar excess over Pol II) and TFIIS (0.1 µM, 5–fold molar excess over Pol II) were added sequentially to test the effect of RECQL5 on the ability of TFIIS to promote read–through of intrinsic blocks to elongation. ( c–f ) Pulse–chase TFIIS read–through assay in the presence of RECQL5 1–620 and RECQL5 IRI performed as indicated in (b). Transcripts were resolved on a denaturing polyacrylamide electrophoresis gel and visualized by phosphorimaging. TFIIS stimulates read–through of three distinct blocks to elongation (TIa, TIb, and TII) to produce a run–off transcript (RO). ( d, f ) Quantitation of the relative amount of transcripts stalled at the TIa site. Error bars denote standard error of the mean (n=3).
Figure Legend Snippet: RECQL5 IRI domain competes with TFIIS for binding to Pol II and inhibits TFIIS–mediated read–through of intrinsic elongation blocks ( a ) Pull-down assay probing Pol II binding to GST–TFIIS in the presence of either wild–type or K598E mutant RECQL5 IRI domains. Bound proteins were analyzed by SDS–PAGE, followed by staining with Coomassie Blue or Western blotting using an anti–Rbp1 antibody. ( b ) Schematic overview of the pulse–chase TFIIS read–through transcription assay using histone H3.3 intron DNA template. Following addition of chase buffer, RECQL5 variants (0.1 µM, 1.0 µM and 10 µM final concentration, corresponding to 5–, 50– and 500–fold molar excess over Pol II) and TFIIS (0.1 µM, 5–fold molar excess over Pol II) were added sequentially to test the effect of RECQL5 on the ability of TFIIS to promote read–through of intrinsic blocks to elongation. ( c–f ) Pulse–chase TFIIS read–through assay in the presence of RECQL5 1–620 and RECQL5 IRI performed as indicated in (b). Transcripts were resolved on a denaturing polyacrylamide electrophoresis gel and visualized by phosphorimaging. TFIIS stimulates read–through of three distinct blocks to elongation (TIa, TIb, and TII) to produce a run–off transcript (RO). ( d, f ) Quantitation of the relative amount of transcripts stalled at the TIa site. Error bars denote standard error of the mean (n=3).

Techniques Used: Binding Assay, Pull Down Assay, Mutagenesis, SDS Page, Staining, Western Blot, Pulse Chase, Concentration Assay, Electrophoresis, Quantitation Assay

6) Product Images from "Bax Forms an Oligomer via Separate, Yet Interdependent, Surfaces *"

Article Title: Bax Forms an Oligomer via Separate, Yet Interdependent, Surfaces *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.113456

Site-specific photocross-linking detected Bax dimerization in Triton X-100. A , photocross-linking of His 6 -tagged Bax to in vitro synthesized [ 35 S]Met-labeled Bax with a single ANB probe attached to K73 ( lanes 1–4 ). The schematic underneath illustrates interaction between the two proteins (indicated by the double-headed arrow ), in which the star attached to the front surface of [ 35 S]Met-Bax indicates the ANB probe. After photolysis the fractions that bound to Ni 2+ -resin were analyzed by SDS-PAGE and phosphorimaging. A photoadduct was detected between the Bax K73 and His 6 -Bax in lane 2 and is indicated by an arrow . The adduct was absent when in vitro synthesized Bax K0 instead of Bax K73 was used ( lanes 5–8 ). The filled circle adjacent to lane 5 indicates a non-photoadduct formed by [ 35 S]Met-Bax K0. The open circle indicates the [ 35 S]Met-labeled Bax K73 or K0 monomer. Protein standards are indicated on the side of each phosphor image with molecular mass. B , inhibition of photocross-linking of the [ 35 S]Met/ANB-labeled Bax K73 to His 6 -tagged Bax by His 6 -tagged Bcl-2ΔTM ( lanes 1–4 ) but not by Bcl-2ΔTM-G145A ( lanes 5–7 ). The arrow indicates the photoadduct of [ 35 S]Met-Bax and His 6 -Bax, and the arrowhead is the photoadduct of [ 35 S]Met-Bax and His 6 -Bcl-2ΔTM or the G145A mutant. These photoadducts are specific to the corresponding protein pairs as they were not detected when one of the proteins ( lanes 2 and 3 ), ϵANB-Lys-tRNA Lys ( lanes 1 and 7 ), or UV irradiation (data not shown) was omitted. The schematic underneath illustrates that His 6 -tagged Bax, and His 6 -tagged Bcl-2ΔTM compete for interaction with [ 35 S]Met- and ANB-labeled Bax. Other symbols are as described in panel A .
Figure Legend Snippet: Site-specific photocross-linking detected Bax dimerization in Triton X-100. A , photocross-linking of His 6 -tagged Bax to in vitro synthesized [ 35 S]Met-labeled Bax with a single ANB probe attached to K73 ( lanes 1–4 ). The schematic underneath illustrates interaction between the two proteins (indicated by the double-headed arrow ), in which the star attached to the front surface of [ 35 S]Met-Bax indicates the ANB probe. After photolysis the fractions that bound to Ni 2+ -resin were analyzed by SDS-PAGE and phosphorimaging. A photoadduct was detected between the Bax K73 and His 6 -Bax in lane 2 and is indicated by an arrow . The adduct was absent when in vitro synthesized Bax K0 instead of Bax K73 was used ( lanes 5–8 ). The filled circle adjacent to lane 5 indicates a non-photoadduct formed by [ 35 S]Met-Bax K0. The open circle indicates the [ 35 S]Met-labeled Bax K73 or K0 monomer. Protein standards are indicated on the side of each phosphor image with molecular mass. B , inhibition of photocross-linking of the [ 35 S]Met/ANB-labeled Bax K73 to His 6 -tagged Bax by His 6 -tagged Bcl-2ΔTM ( lanes 1–4 ) but not by Bcl-2ΔTM-G145A ( lanes 5–7 ). The arrow indicates the photoadduct of [ 35 S]Met-Bax and His 6 -Bax, and the arrowhead is the photoadduct of [ 35 S]Met-Bax and His 6 -Bcl-2ΔTM or the G145A mutant. These photoadducts are specific to the corresponding protein pairs as they were not detected when one of the proteins ( lanes 2 and 3 ), ϵANB-Lys-tRNA Lys ( lanes 1 and 7 ), or UV irradiation (data not shown) was omitted. The schematic underneath illustrates that His 6 -tagged Bax, and His 6 -tagged Bcl-2ΔTM compete for interaction with [ 35 S]Met- and ANB-labeled Bax. Other symbols are as described in panel A .

Techniques Used: In Vitro, Synthesized, Labeling, SDS Page, Inhibition, Mutagenesis, Irradiation

Oligomerization of Bax induced by Triton X-100 and inhibited by Bcl-2. A , oligomerization of in vitro synthesized [ 35 S]Met-labeled Bax ( 35 S-Bax ) and purified His 6 -tagged Bax ( 6H-Bax ) proteins in the absence and presence of Triton X-100 examined by gel-filtration chromatography. The chromatographic fractions 21–33 were analyzed by SDS-PAGE and phosphorimaging or immunoblotting with a Bax-specific antibody to detect [ 35 S]Met-Bax ( top two panels ) or 6H-Bax ( bottom two panels ), respectively. B , oligomerization of Triton X-100-treated His 6 -Bax in the absence or presence of His 6 -tagged Bcl-2ΔTM or Bcl-2ΔTM-G145A examined by gel-filtration chromatography. The chromatographic fractions 21–33 were analyzed by SDS-PAGE and immunoblotting with a Bax- or Bcl-2-specific antibody to detect His 6 -tagged Bax ( left panels ) or Bcl-2 ( right panels ). The molar ratios of Bax versus Bcl-2 or Bcl-2-G145A in the samples are indicated in the middle . In all panels , the elution positions for protein standards are indicated on the top with molecular mass.
Figure Legend Snippet: Oligomerization of Bax induced by Triton X-100 and inhibited by Bcl-2. A , oligomerization of in vitro synthesized [ 35 S]Met-labeled Bax ( 35 S-Bax ) and purified His 6 -tagged Bax ( 6H-Bax ) proteins in the absence and presence of Triton X-100 examined by gel-filtration chromatography. The chromatographic fractions 21–33 were analyzed by SDS-PAGE and phosphorimaging or immunoblotting with a Bax-specific antibody to detect [ 35 S]Met-Bax ( top two panels ) or 6H-Bax ( bottom two panels ), respectively. B , oligomerization of Triton X-100-treated His 6 -Bax in the absence or presence of His 6 -tagged Bcl-2ΔTM or Bcl-2ΔTM-G145A examined by gel-filtration chromatography. The chromatographic fractions 21–33 were analyzed by SDS-PAGE and immunoblotting with a Bax- or Bcl-2-specific antibody to detect His 6 -tagged Bax ( left panels ) or Bcl-2 ( right panels ). The molar ratios of Bax versus Bcl-2 or Bcl-2-G145A in the samples are indicated in the middle . In all panels , the elution positions for protein standards are indicated on the top with molecular mass.

Techniques Used: In Vitro, Synthesized, Labeling, Purification, Filtration, Chromatography, SDS Page

7) Product Images from "Inhibition of a Golgi Complex Lysophospholipid Acyltransferase Induces Membrane Tubule Formation and Retrograde Trafficking"

Article Title: Inhibition of a Golgi Complex Lysophospholipid Acyltransferase Induces Membrane Tubule Formation and Retrograde Trafficking

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E02-11-0711

Golgi-associated, CI-976-sensitive LPAT activity uses LPC, but not LPA, as the acyl-chain acceptor. The conversion of LPL to PL was monitored by incubating isolated Golgi membranes with various LPA or LPC substrates, CI-976 (50 μM), and [ 14 C]palmitoyl-CoA at 37°C for 5 min. Total lipids were extracted, separated by TLC and the radioactivity of PA- or PC-containing spots was measured by phosphorimaging. The enzyme activity is reported as the percentage of the control (mean plus 1 SD from three experiments).
Figure Legend Snippet: Golgi-associated, CI-976-sensitive LPAT activity uses LPC, but not LPA, as the acyl-chain acceptor. The conversion of LPL to PL was monitored by incubating isolated Golgi membranes with various LPA or LPC substrates, CI-976 (50 μM), and [ 14 C]palmitoyl-CoA at 37°C for 5 min. Total lipids were extracted, separated by TLC and the radioactivity of PA- or PC-containing spots was measured by phosphorimaging. The enzyme activity is reported as the percentage of the control (mean plus 1 SD from three experiments).

Techniques Used: Activity Assay, Isolation, Thin Layer Chromatography, Radioactivity

8) Product Images from "Assaying kinase activity of the TPL-2/NF-κB1 p105/ABIN-2 complex using an optimal peptide substrate"

Article Title: Assaying kinase activity of the TPL-2/NF-κB1 p105/ABIN-2 complex using an optimal peptide substrate

Journal: Biochemical Journal

doi: 10.1042/BCJ20170579

Testing an optimized peptide substrate for TPL-2/NF-κB1 p105/ABIN-2. ( A ) The primary and secondary amino acid preferences for phosphorylation by the recombinant TPL-2/NF-κB1 p105/ABIN-2 complex are shown. ( B ) The sequences and scansite scores for the MKK1 activation loop and optimized TPL-2tide peptide substrates. ( C ) Time-course experiment comparing TPL-2/NF-κB1 p105/ABIN-2 complex phosphorylation of MKK1 and TPL-2tide peptides (50 µM final concentration). Assays were performed with 30 nM the recombinant TPL-2/NF-κB1 p105/ABIN-2 complex in TPL-2 kinase buffer plus 1 mM ATP and 0.02 µCi/µL [γ- 32 P]ATP. Peptides were transferred onto streptavidin-coated membranes, which were then extensively washed. Incorporation of 32 P into peptides was quantified by phosphorimaging. Linear regression was fitted with GraFit version 7.0.3. Values are means ± SD for three replicate reactions. ( D ) Kinase assays as in ( C ) comparing TPL-2tide peptide phosphorylation by wild-type (WT) or kinase-inactive (D270A) TPL-2/NF-κB1 p105/ABIN-2 complex (15 min). ( E and F ) TPL-2 30–404 and TPL-2/NF-κB1 p105/ABIN-2 were titrated (1 : 1.66) in 384-well plates. A substrate solution containing ATP and TPL-2tide ( E ) or S5 peptide ( F ) was then added. The plates were analyzed using a Sciex API6500 ( E ) or API4000 ( F ) Triple Quad with RapidFire™ Technology. The graph shows total peak area counts for the MRM transition 953.6/904.3 Da ( E ; phosphorylated TPL-2tide peptide) and 612.9/120 Da ( F ; phosphorylated S5 peptide) for the indicated enzyme at increasing concentrations. Linear regression was fitted using GraFit software. In E and F , representative graphs from one experiment are shown, including standard deviation of triplicate assays.
Figure Legend Snippet: Testing an optimized peptide substrate for TPL-2/NF-κB1 p105/ABIN-2. ( A ) The primary and secondary amino acid preferences for phosphorylation by the recombinant TPL-2/NF-κB1 p105/ABIN-2 complex are shown. ( B ) The sequences and scansite scores for the MKK1 activation loop and optimized TPL-2tide peptide substrates. ( C ) Time-course experiment comparing TPL-2/NF-κB1 p105/ABIN-2 complex phosphorylation of MKK1 and TPL-2tide peptides (50 µM final concentration). Assays were performed with 30 nM the recombinant TPL-2/NF-κB1 p105/ABIN-2 complex in TPL-2 kinase buffer plus 1 mM ATP and 0.02 µCi/µL [γ- 32 P]ATP. Peptides were transferred onto streptavidin-coated membranes, which were then extensively washed. Incorporation of 32 P into peptides was quantified by phosphorimaging. Linear regression was fitted with GraFit version 7.0.3. Values are means ± SD for three replicate reactions. ( D ) Kinase assays as in ( C ) comparing TPL-2tide peptide phosphorylation by wild-type (WT) or kinase-inactive (D270A) TPL-2/NF-κB1 p105/ABIN-2 complex (15 min). ( E and F ) TPL-2 30–404 and TPL-2/NF-κB1 p105/ABIN-2 were titrated (1 : 1.66) in 384-well plates. A substrate solution containing ATP and TPL-2tide ( E ) or S5 peptide ( F ) was then added. The plates were analyzed using a Sciex API6500 ( E ) or API4000 ( F ) Triple Quad with RapidFire™ Technology. The graph shows total peak area counts for the MRM transition 953.6/904.3 Da ( E ; phosphorylated TPL-2tide peptide) and 612.9/120 Da ( F ; phosphorylated S5 peptide) for the indicated enzyme at increasing concentrations. Linear regression was fitted using GraFit software. In E and F , representative graphs from one experiment are shown, including standard deviation of triplicate assays.

Techniques Used: Recombinant, Activation Assay, Concentration Assay, Software, Standard Deviation

The primary amino acid sequence specificity of TPL-2. The peptide library comprised 198 individual biotinylated peptide mixtures. Each peptide contained a central phosphoracceptor Ser or Thr, flanked by degenerate positions, comprising an equimolar mixture of the 17 amino acids, excluding Cys, Ser and Thr. In each peptide, one position was fixed (fixed residue) with one of the 20 naturally occurring unmodified amino acids, phosphor-Thr (pT) or phosphor-Tyr (pY). Peptides were incubated with 30 nM recombinant TPL-2/NF-κB1 p105/ABIN-2 complex at a final substrate concentration of 50 µM at 30°C for 1 h. Assays were performed in TPL-2 kinase buffer plus 10 µM ATP and 3 nCi/µL [γ- 32 P]ATP in the absence ( A ) or presence ( B ) of C34 TPL-2 inhibitor (10 µM). Following incubation, reactions were transferred onto SAM2 membranes, which were then washed extensively. Incorporation of 32 P into peptides was quantified by phosphorimaging.
Figure Legend Snippet: The primary amino acid sequence specificity of TPL-2. The peptide library comprised 198 individual biotinylated peptide mixtures. Each peptide contained a central phosphoracceptor Ser or Thr, flanked by degenerate positions, comprising an equimolar mixture of the 17 amino acids, excluding Cys, Ser and Thr. In each peptide, one position was fixed (fixed residue) with one of the 20 naturally occurring unmodified amino acids, phosphor-Thr (pT) or phosphor-Tyr (pY). Peptides were incubated with 30 nM recombinant TPL-2/NF-κB1 p105/ABIN-2 complex at a final substrate concentration of 50 µM at 30°C for 1 h. Assays were performed in TPL-2 kinase buffer plus 10 µM ATP and 3 nCi/µL [γ- 32 P]ATP in the absence ( A ) or presence ( B ) of C34 TPL-2 inhibitor (10 µM). Following incubation, reactions were transferred onto SAM2 membranes, which were then washed extensively. Incorporation of 32 P into peptides was quantified by phosphorimaging.

Techniques Used: Sequencing, Incubation, Recombinant, Concentration Assay

9) Product Images from "Polyketide Chain Skipping Mechanism in the Biosynthesis of the Hybrid Nonribosomal Peptide-Polyketide Antitumor Antibiotic Leinamycin in Streptomyces atroolivaceus S-140 ⊥"

Article Title: Polyketide Chain Skipping Mechanism in the Biosynthesis of the Hybrid Nonribosomal Peptide-Polyketide Antitumor Antibiotic Leinamycin in Streptomyces atroolivaceus S-140 ⊥

Journal:

doi: 10.1021/np050467t

In vitro assays of LnmG-catalyzed loading of malonyl CoA to LnmJ ACP 6-1 and ACP 6-2 . (A) Incubation of ACP 6-1 and ACP 6-2 with [2- 14 C]malonyl CoA and LnmG as visualized on a 4– 15 % SDSPAGE (I) and by phosphorimaging (II). Lane 1, molecular weight
Figure Legend Snippet: In vitro assays of LnmG-catalyzed loading of malonyl CoA to LnmJ ACP 6-1 and ACP 6-2 . (A) Incubation of ACP 6-1 and ACP 6-2 with [2- 14 C]malonyl CoA and LnmG as visualized on a 4– 15 % SDSPAGE (I) and by phosphorimaging (II). Lane 1, molecular weight

Techniques Used: In Vitro, Incubation, Molecular Weight

10) Product Images from "A novel mechanism for regulating the activity of proliferating cell nuclear antigen by a small protein"

Article Title: A novel mechanism for regulating the activity of proliferating cell nuclear antigen by a small protein

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku239

TIP inhibits PCNA stimulation of Fen1 activity. ( A ) A schematic illustration of the assay. ( B and C ) The effect of PCNA on Fen1 activity was measured as described in ‘Materials and Methods’ section in a reaction mixture (20 μl) that contained 20 fmol substrates, 250 fmol Fen1 (lanes 1–5 and 7–11), 188 fmol (lane 2), 375 fmol (lane 3), 750 fmol (lane 4), or 1500 fmol (lanes 5 and 6) of PCNA1, or 188 fmol (lane 8), 375 fmol (lane 9), 750 fmol (lane 10), or 1500 fmol (lanes 11 and 12) of PCNA2. The reactions mixtures were incubated at 60°C for 60 min, and the products were separated on 20% (w/v) polyacrylamide–8 M urea gels, visualized and quantified by phosphorimaging. A representative gel is shown in panel (B), and the average, with standard deviation, from three independent experiments are shown in panel (C). ( D and E ) The effect of TIP on PCNA stimulation of Fen1 activity was measured as described in ‘Materials and Methods’ section in a reaction mixture (20 μl) that contained 20 fmol substrates, 250 fmol Fen1, 1500 fmol PCNA1 (lanes 2–7), or 1500 fmol PCNA2 (lanes 9–14) in the presence of 375 fmol (lanes 3 and 10), 750 fmol (lanes 4 and 11), 1500 fmol (lanes 5 and 12), 3000 fmol (lanes 6 and 13), or 6000 fmol (lanes 7 and 14) of TIP. The reaction mixtures were incubated at 60°C for 60 min and the products separated on 20% (w/v) polyacrylamide–8 M urea gels, visualized and quantified by phosphorimaging. A representative gel is shown in panel (D) and the average, with standard deviations from three independent experiments, are shown in panel (E).
Figure Legend Snippet: TIP inhibits PCNA stimulation of Fen1 activity. ( A ) A schematic illustration of the assay. ( B and C ) The effect of PCNA on Fen1 activity was measured as described in ‘Materials and Methods’ section in a reaction mixture (20 μl) that contained 20 fmol substrates, 250 fmol Fen1 (lanes 1–5 and 7–11), 188 fmol (lane 2), 375 fmol (lane 3), 750 fmol (lane 4), or 1500 fmol (lanes 5 and 6) of PCNA1, or 188 fmol (lane 8), 375 fmol (lane 9), 750 fmol (lane 10), or 1500 fmol (lanes 11 and 12) of PCNA2. The reactions mixtures were incubated at 60°C for 60 min, and the products were separated on 20% (w/v) polyacrylamide–8 M urea gels, visualized and quantified by phosphorimaging. A representative gel is shown in panel (B), and the average, with standard deviation, from three independent experiments are shown in panel (C). ( D and E ) The effect of TIP on PCNA stimulation of Fen1 activity was measured as described in ‘Materials and Methods’ section in a reaction mixture (20 μl) that contained 20 fmol substrates, 250 fmol Fen1, 1500 fmol PCNA1 (lanes 2–7), or 1500 fmol PCNA2 (lanes 9–14) in the presence of 375 fmol (lanes 3 and 10), 750 fmol (lanes 4 and 11), 1500 fmol (lanes 5 and 12), 3000 fmol (lanes 6 and 13), or 6000 fmol (lanes 7 and 14) of TIP. The reaction mixtures were incubated at 60°C for 60 min and the products separated on 20% (w/v) polyacrylamide–8 M urea gels, visualized and quantified by phosphorimaging. A representative gel is shown in panel (D) and the average, with standard deviations from three independent experiments, are shown in panel (E).

Techniques Used: Activity Assay, Incubation, Standard Deviation

Effect of TIP on the stimulation of Fen1 activity by mutant PCNAs. ( A ) A schematic illustration of the assay. ( B ) The residue mutated in PCNA2-A and PCNA2-E. ( C ) The effect of TIP on the stimulation of Fen1 activity by PCNA wild-type and mutant PCNAs was measured as described in ‘Materials and Methods’ section in a reaction mixture (20 μl) that contained 20 fmol substrates, 250 fmol Fen1, 1500 fmol PCNA2 (lanes 3–8), PCNA2-A (lanes 9–14), PCNA2-E (lanes 15–20), in the presence of 375 fmol (lanes 4, 10 and 16), 750 fmol (lanes 5, 11 and 17), 1500 fmol (lanes 6, 12 and 18), 3000 fmol (lanes 7, 13 and 19), or 6000 fmol (lanes 8, 14 and 20) of TIP. Oligonucleotides of 49 nt and 13 nt were separated in lane 1 and marked as ‘S’ and ‘P’, respectively. Reaction mixtures were incubated at 60°C for 60 min and the products separated on 10% (w/v) polyacrylamide–8 M urea gels, visualized and quantified by phosphorimaging. A representative gel is shown in panel ( C ) and the averages with standard deviations from three independent experiments are shown in panel ( D ).
Figure Legend Snippet: Effect of TIP on the stimulation of Fen1 activity by mutant PCNAs. ( A ) A schematic illustration of the assay. ( B ) The residue mutated in PCNA2-A and PCNA2-E. ( C ) The effect of TIP on the stimulation of Fen1 activity by PCNA wild-type and mutant PCNAs was measured as described in ‘Materials and Methods’ section in a reaction mixture (20 μl) that contained 20 fmol substrates, 250 fmol Fen1, 1500 fmol PCNA2 (lanes 3–8), PCNA2-A (lanes 9–14), PCNA2-E (lanes 15–20), in the presence of 375 fmol (lanes 4, 10 and 16), 750 fmol (lanes 5, 11 and 17), 1500 fmol (lanes 6, 12 and 18), 3000 fmol (lanes 7, 13 and 19), or 6000 fmol (lanes 8, 14 and 20) of TIP. Oligonucleotides of 49 nt and 13 nt were separated in lane 1 and marked as ‘S’ and ‘P’, respectively. Reaction mixtures were incubated at 60°C for 60 min and the products separated on 10% (w/v) polyacrylamide–8 M urea gels, visualized and quantified by phosphorimaging. A representative gel is shown in panel ( C ) and the averages with standard deviations from three independent experiments are shown in panel ( D ).

Techniques Used: Activity Assay, Mutagenesis, Incubation

11) Product Images from "Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope"

Article Title: Distinct Pathways Mediate the Sorting of Tail-Anchored Proteins to the Plastid Outer Envelope

Journal: PLoS ONE

doi: 10.1371/journal.pone.0010098

OEP9, Toc33 and Toc34 insert into trypsin-pretreated chloroplasts and ppi1 and ppi3 chloroplasts in vitro . (a) Insertion of OEP9, Toc33, Toc34 and SSU into trypsin-pretreated chloroplasts in vitro . Isolated Arabidopsis chloroplasts pre-treated with trypsin were incubated with in vitro synthesized translation products (TP) including either myc-OEP9, myc-Toc33, myc-Toc34 or SSU and then resuspended with Na 2 CO 3 (see ‘ Materials and Methods ’ for details). Equivalent amounts of Na 2 CO 3 - or mock-extracted chloroplast membranes were then subjected to SDS-PAGE/phosphorimaging. Addition of Na 2 CO 3 to the reaction mixtures is indicated as (+), omission as (−). The migration in the gel of each protein is marked with an arrowhead to the right of each panel. (b) Insertion of OEP9, Toc33, Toc34 and SSU into ppi1 and ppi3 chloroplasts in vitro . Chloroplasts isolated from ppi1 or ppi3 mutant Arabidopsis plants were incubated with in vitro synthesized TP including either myc-tagged OEP9, myc-Toc33 myc-Toc34 or SSU. Chloroplasts were then resuspended with or without Na 2 CO 3 and subjected to SDS-PAGE/phosophorimaging. Addition of Na 2 CO 3 to the reaction mixtures is indicated as (+), omission as (−). The migration in the gel of each (full-length) protein is marked with a solid arrowhead to the right of each panel, whereas the mature, processed (cleaved) form of SSU, is indicated with open arrowhead. Note that the smaller, additional bands observed in some of the myc-tagged OEP9, Toc33 and Toc34 lanes (e.g., lane 1) were present in varied amounts depending on the translation reaction (cf. lane 1 here and lane 1 in Figure 3 ) and are, as described previously for Toc33 [27] and psToc34 [24] , likely truncated versions of these proteins due to internal translation initiation(s).
Figure Legend Snippet: OEP9, Toc33 and Toc34 insert into trypsin-pretreated chloroplasts and ppi1 and ppi3 chloroplasts in vitro . (a) Insertion of OEP9, Toc33, Toc34 and SSU into trypsin-pretreated chloroplasts in vitro . Isolated Arabidopsis chloroplasts pre-treated with trypsin were incubated with in vitro synthesized translation products (TP) including either myc-OEP9, myc-Toc33, myc-Toc34 or SSU and then resuspended with Na 2 CO 3 (see ‘ Materials and Methods ’ for details). Equivalent amounts of Na 2 CO 3 - or mock-extracted chloroplast membranes were then subjected to SDS-PAGE/phosphorimaging. Addition of Na 2 CO 3 to the reaction mixtures is indicated as (+), omission as (−). The migration in the gel of each protein is marked with an arrowhead to the right of each panel. (b) Insertion of OEP9, Toc33, Toc34 and SSU into ppi1 and ppi3 chloroplasts in vitro . Chloroplasts isolated from ppi1 or ppi3 mutant Arabidopsis plants were incubated with in vitro synthesized TP including either myc-tagged OEP9, myc-Toc33 myc-Toc34 or SSU. Chloroplasts were then resuspended with or without Na 2 CO 3 and subjected to SDS-PAGE/phosophorimaging. Addition of Na 2 CO 3 to the reaction mixtures is indicated as (+), omission as (−). The migration in the gel of each (full-length) protein is marked with a solid arrowhead to the right of each panel, whereas the mature, processed (cleaved) form of SSU, is indicated with open arrowhead. Note that the smaller, additional bands observed in some of the myc-tagged OEP9, Toc33 and Toc34 lanes (e.g., lane 1) were present in varied amounts depending on the translation reaction (cf. lane 1 here and lane 1 in Figure 3 ) and are, as described previously for Toc33 [27] and psToc34 [24] , likely truncated versions of these proteins due to internal translation initiation(s).

Techniques Used: In Vitro, Isolation, Incubation, Synthesized, SDS Page, Migration, Mutagenesis

12) Product Images from "Thrombopoietin-Mediated Sustained Activation of Extracellular Signal-Regulated Kinase in UT7-Mpl Cells Requires Both Ras-Raf-1- and Rap1-B-Raf-Dependent Pathways"

Article Title: Thrombopoietin-Mediated Sustained Activation of Extracellular Signal-Regulated Kinase in UT7-Mpl Cells Requires Both Ras-Raf-1- and Rap1-B-Raf-Dependent Pathways

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.8.2659-2670.2001

In UT7-Mpl cells, both Ras and Rap1 are required for TPO-induced Elk1 and ERK activation. (A) Inhibition of Elk1 transcriptional activity by expression of interfering Ras and Rap1 mutants. UT7-Mpl cells were transfected with 5 μg of pFA-Elk1 and 10 μg of pFR-luc plasmids of the PathDetect reporting system, along with 1 μg of pcDNA encoding β-galactosidase and 10 μg of either empty vectors or plasmids encoding RasN17, Rap1N17, or Spa1, as indicated. Cells were starved of cytokine overnight and treated for 24 h with TPO before being harvested. Luciferase activity was measured as described in Materials and Methods and normalized to β-galactosidase activity. Results are expressed as fold induction of luciferase activity over that induced in cells transfected with empty pcDNA alone. Average values from eight independent experiments are presented with SEs indicated by the error bars. (B) Rap1 is required for the sustained activation of ERK in response to TPO. UT7-Mpl cells were transiently transfected with plasmid encoding 10 μg of HA-tagged ERK1 along with 10 μg of empty pcDNA or pcDNA encoding RasN17 and Rap1N17, as indicated. After overnight starvation of cytokines, cells were left untreated or stimulated with 10 nM TPO for 1 or 7 h. Transfected ERK was immunoprecipitated with anti-HA monoclonal antibody and subjected to immune kinase assay using MBP as a substrate. Radioactivity was quantified by phosphorimaging and was plotted as relative ERK activity. The expression of equal levels of transfected HA-ERK1 was confirmed by Western blotting (WB) with anti-HA antibody.
Figure Legend Snippet: In UT7-Mpl cells, both Ras and Rap1 are required for TPO-induced Elk1 and ERK activation. (A) Inhibition of Elk1 transcriptional activity by expression of interfering Ras and Rap1 mutants. UT7-Mpl cells were transfected with 5 μg of pFA-Elk1 and 10 μg of pFR-luc plasmids of the PathDetect reporting system, along with 1 μg of pcDNA encoding β-galactosidase and 10 μg of either empty vectors or plasmids encoding RasN17, Rap1N17, or Spa1, as indicated. Cells were starved of cytokine overnight and treated for 24 h with TPO before being harvested. Luciferase activity was measured as described in Materials and Methods and normalized to β-galactosidase activity. Results are expressed as fold induction of luciferase activity over that induced in cells transfected with empty pcDNA alone. Average values from eight independent experiments are presented with SEs indicated by the error bars. (B) Rap1 is required for the sustained activation of ERK in response to TPO. UT7-Mpl cells were transiently transfected with plasmid encoding 10 μg of HA-tagged ERK1 along with 10 μg of empty pcDNA or pcDNA encoding RasN17 and Rap1N17, as indicated. After overnight starvation of cytokines, cells were left untreated or stimulated with 10 nM TPO for 1 or 7 h. Transfected ERK was immunoprecipitated with anti-HA monoclonal antibody and subjected to immune kinase assay using MBP as a substrate. Radioactivity was quantified by phosphorimaging and was plotted as relative ERK activity. The expression of equal levels of transfected HA-ERK1 was confirmed by Western blotting (WB) with anti-HA antibody.

Techniques Used: Activation Assay, Inhibition, Activity Assay, Expressing, Transfection, Luciferase, Plasmid Preparation, Immunoprecipitation, Kinase Assay, Radioactivity, Western Blot

13) Product Images from "Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming"

Article Title: Few basepairing-independent motifs in the apical half of the avian HBV ε RNA stem-loop determine site-specific initiation of protein-priming

Journal: Scientific Reports

doi: 10.1038/s41598-017-07657-z

Interrogating a potential space-bar function of the upper Dε stem. ( A ) Sequence alterations in class I and class II variants. The distance between bulge and loop in the replication-proficient Dε variant S12 22 was altered by deleting two of its non-authentic residues on the left or right upper half-stem or both, or by analogously introducing two extra C residues on one or both sides (class I). The same changes were introduced in a variant lacking a priori six nt in the lower right half-stem (class II). The encircled C represents the dominant initiation site. ( B ) In vitro priming activities. The indicated Dε variants were in vitro transcribed and subjected to α 32 P-dGTP priming assays using full-length DHBV polymerase in rabbit reticulocyte lysate (RRL), or recombinant miniDP protein. 32 P-labeled P protein was visualized, after SDS-PAGE, by autoradiography. Signal intensities were quantified by phosphorimaging; numbers below each lane show the mean relative priming signals ± standard deviation (SD; n ≥ 3) compared to wt Dε RNA which was set to 100%; nd, not detectable. Analogous data for α 32 P-dATP are shown in Supplementary Fig. S1 . ( C ) No impact of typical class I and class II mutations on dNTP specificity during in vitro priming. MiniDP priming assays were performed using either α 32 P-dGTP or α 32 P-dATP in the presence of only Mg 2+ , or Mg 2+ plus Mn 2+ . In either constellation, dGTP was incorporated ~20-fold more efficiently than dATP. ( D ) Class I and class II variants support viral replication. LMH cells were transfected with pCD16 vectors bearing the indicated mutant sequences in both 5′ and 3′ Dε. Vectors encoding wt-DHBV16 (wt), a variant defective in 5′ Dε (DHBV Δε), and the upper stem variants S5 and S12 22 , 30 served as controls. DNA from cytoplasmic nucleocapsids was analyzed by Southern blotting using a 32 P-labeled DHBV DNA probe. Numbers indicate mean signal intensities ± SD (n ≥ 3) of full-length DNAs (RC + dsL) relative to wt-DHBV which was set to 100%.
Figure Legend Snippet: Interrogating a potential space-bar function of the upper Dε stem. ( A ) Sequence alterations in class I and class II variants. The distance between bulge and loop in the replication-proficient Dε variant S12 22 was altered by deleting two of its non-authentic residues on the left or right upper half-stem or both, or by analogously introducing two extra C residues on one or both sides (class I). The same changes were introduced in a variant lacking a priori six nt in the lower right half-stem (class II). The encircled C represents the dominant initiation site. ( B ) In vitro priming activities. The indicated Dε variants were in vitro transcribed and subjected to α 32 P-dGTP priming assays using full-length DHBV polymerase in rabbit reticulocyte lysate (RRL), or recombinant miniDP protein. 32 P-labeled P protein was visualized, after SDS-PAGE, by autoradiography. Signal intensities were quantified by phosphorimaging; numbers below each lane show the mean relative priming signals ± standard deviation (SD; n ≥ 3) compared to wt Dε RNA which was set to 100%; nd, not detectable. Analogous data for α 32 P-dATP are shown in Supplementary Fig. S1 . ( C ) No impact of typical class I and class II mutations on dNTP specificity during in vitro priming. MiniDP priming assays were performed using either α 32 P-dGTP or α 32 P-dATP in the presence of only Mg 2+ , or Mg 2+ plus Mn 2+ . In either constellation, dGTP was incorporated ~20-fold more efficiently than dATP. ( D ) Class I and class II variants support viral replication. LMH cells were transfected with pCD16 vectors bearing the indicated mutant sequences in both 5′ and 3′ Dε. Vectors encoding wt-DHBV16 (wt), a variant defective in 5′ Dε (DHBV Δε), and the upper stem variants S5 and S12 22 , 30 served as controls. DNA from cytoplasmic nucleocapsids was analyzed by Southern blotting using a 32 P-labeled DHBV DNA probe. Numbers indicate mean signal intensities ± SD (n ≥ 3) of full-length DNAs (RC + dsL) relative to wt-DHBV which was set to 100%.

Techniques Used: Sequencing, Variant Assay, In Vitro, Recombinant, Labeling, SDS Page, Autoradiography, Standard Deviation, Transfection, Mutagenesis, Southern Blot

Impact of bulge region architecture on initiation site selection. Dε RNAs in which the unpaired U opposite the bulge (uA, uG, uC, or uΔ where the U was deleted) and the initiation site C at the b6 position (b6G, b6A, b6U) were mutated individually or in combination, were analyzed by miniDP priming assays with either of the four dNTPs. Variants are designated as uXb6Y, with X and Y defining the nt at the unpaired U and the b6 position, respectively. ( A ) Autoradiograms of individual priming assays. Note the preference of all substantially active variants for the nt complementary to the respective b6 position, except those with b6G which equally utilized dTTP and dCTP. ( B ) Graphical representation. Signal intensities were determined by phosphorimaging and related to the dGTP priming signal with wt Dε (uUb6C) which was set to 100%. Note the strong reduction in overall priming efficiency for all variants carrying a G at the unpaired U position, and the preference for utilizing the dNTP complementary to b6, except for b6G. ( C ) Schematic correlation of priming efficacy and b6 initiation site specificity with structural impact on the bulge region. RNA variants were categorized for overall priming efficacy and preference for utilizing the dNTP complementary to b6. Structural impact was assessed by the potential for new canonical plus G-U pairs. A high potential correlated with low overall priming; assessments for all variants are shown in Supplementary Fig. S5 . The relaxed specificity of uUb6G and uAb6G for both dCTP and dTTP is in line with the importance of a G following the initiation site (see Fig. 6 and below). ( D ) Impact of the top bulge closing nt on priming efficacy and initiation site selection. The G-C bulge closing G-C pair in wt-Dε was swapped to c-g. Both RNAs were analyzed in parallel in miniDP priming assays with all four dNTPs. Note the drastic drop in priming efficacy but detectable maintainance of dGTP preference in the mutant. ( E ) Impact of the nt opposite the G following the bulge. Priming capacity was assessed by miniDP assays with dGTP. Note the ample options for new basepairings in mutants G-g and G-a.
Figure Legend Snippet: Impact of bulge region architecture on initiation site selection. Dε RNAs in which the unpaired U opposite the bulge (uA, uG, uC, or uΔ where the U was deleted) and the initiation site C at the b6 position (b6G, b6A, b6U) were mutated individually or in combination, were analyzed by miniDP priming assays with either of the four dNTPs. Variants are designated as uXb6Y, with X and Y defining the nt at the unpaired U and the b6 position, respectively. ( A ) Autoradiograms of individual priming assays. Note the preference of all substantially active variants for the nt complementary to the respective b6 position, except those with b6G which equally utilized dTTP and dCTP. ( B ) Graphical representation. Signal intensities were determined by phosphorimaging and related to the dGTP priming signal with wt Dε (uUb6C) which was set to 100%. Note the strong reduction in overall priming efficiency for all variants carrying a G at the unpaired U position, and the preference for utilizing the dNTP complementary to b6, except for b6G. ( C ) Schematic correlation of priming efficacy and b6 initiation site specificity with structural impact on the bulge region. RNA variants were categorized for overall priming efficacy and preference for utilizing the dNTP complementary to b6. Structural impact was assessed by the potential for new canonical plus G-U pairs. A high potential correlated with low overall priming; assessments for all variants are shown in Supplementary Fig. S5 . The relaxed specificity of uUb6G and uAb6G for both dCTP and dTTP is in line with the importance of a G following the initiation site (see Fig. 6 and below). ( D ) Impact of the top bulge closing nt on priming efficacy and initiation site selection. The G-C bulge closing G-C pair in wt-Dε was swapped to c-g. Both RNAs were analyzed in parallel in miniDP priming assays with all four dNTPs. Note the drastic drop in priming efficacy but detectable maintainance of dGTP preference in the mutant. ( E ) Impact of the nt opposite the G following the bulge. Priming capacity was assessed by miniDP assays with dGTP. Note the ample options for new basepairings in mutants G-g and G-a.

Techniques Used: Selection, Mutagenesis

The GUUGU motif and the G following the bulge maintain functionality in the absence of a defined structure context. ( A ) Sequences of mini-Dε1 and mini-Dε2 RNAs. The two U residues distinguishing mini-Dε2 from mini-Dε1 are encircled. Lower case lettering indicates non-wt nt; those shown in orange in the lower stem were present in the RNAs used for in vitro priming but not in the mini-Dε pCD16_Δ3′ε vectors used to assess replication competence. ( B ) Mini-Dε in vitro priming activity. In vitro transcribed mini-Dε RNAs were subjected to miniDP priming assays with dGTP, in parallel with controls without RNA (ø) and with wt-Dε RNA; of the latter sample, only half as much of the reaction was loaded (1/2 wt). Signals were quantified by phosphorimaging and those of the variants were related to twice the intensity (set as 100%) of the 1/2 wt signal. ( C ) Mini-Dε1 and mini-Dε2 support DHBV replication. LMH cells were transfected with wt-DHBV vector pCD16_Δ3′ε or derivatives carrying mini-Dε1 and mini-Dε2 at the 5′ ε position. DNA from cytoplasmic nucleocapsids was analyzed by Southern blotting using a 32 P-labeled DHBV DNA probe.
Figure Legend Snippet: The GUUGU motif and the G following the bulge maintain functionality in the absence of a defined structure context. ( A ) Sequences of mini-Dε1 and mini-Dε2 RNAs. The two U residues distinguishing mini-Dε2 from mini-Dε1 are encircled. Lower case lettering indicates non-wt nt; those shown in orange in the lower stem were present in the RNAs used for in vitro priming but not in the mini-Dε pCD16_Δ3′ε vectors used to assess replication competence. ( B ) Mini-Dε in vitro priming activity. In vitro transcribed mini-Dε RNAs were subjected to miniDP priming assays with dGTP, in parallel with controls without RNA (ø) and with wt-Dε RNA; of the latter sample, only half as much of the reaction was loaded (1/2 wt). Signals were quantified by phosphorimaging and those of the variants were related to twice the intensity (set as 100%) of the 1/2 wt signal. ( C ) Mini-Dε1 and mini-Dε2 support DHBV replication. LMH cells were transfected with wt-DHBV vector pCD16_Δ3′ε or derivatives carrying mini-Dε1 and mini-Dε2 at the 5′ ε position. DNA from cytoplasmic nucleocapsids was analyzed by Southern blotting using a 32 P-labeled DHBV DNA probe.

Techniques Used: In Vitro, Activity Assay, Transfection, Plasmid Preparation, Southern Blot, Labeling

14) Product Images from "Functional Evolution in Orthologous Cell-encoded RNA-dependent RNA Polymerases *"

Article Title: Functional Evolution in Orthologous Cell-encoded RNA-dependent RNA Polymerases *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.685933

QDE-1 orthologs have distinct enzymatic properties. A and B , RNA polymerase activities of purified catalytic fragments of QDE-1 Ncr and QDE-1 Tte were assayed at 30–60 °C in the presence of an ssRNA ( A ) or an ssDNA template ( B ). The reaction products were separated by native agarose gel electrophoresis and visualized using ethidium bromide staining or 32 P phosphorimaging, as indicated. Note that QDE-1 Tte is substantially more efficient than QDE-1 Ncr in generating sRNA products that migrate either at a low molecular weight position or in a template base-paired form. C , RNA polymerase activities of QDE-1 Ncr and QDE-1 Tte were assayed at 45 °C in the presence of a ssRNA template and analyzed by denaturing agarose gel electrophoresis. D , RNA products from C were incubated with increasing concentrations of RNase ONE or RNase ONE reaction buffer, as specified under “Experimental Procedures.” Positions of the 1× full template-length products of processive end-to-end polymerization initiated in a primer-independent manner and sRNA products of non-processive polymerization are indicated on the right . Also shown is an expected position of “back-primed” 2× template-length products, which QDE-1 Ncr can generate for some but not all ssRNA templates ( 33 ).
Figure Legend Snippet: QDE-1 orthologs have distinct enzymatic properties. A and B , RNA polymerase activities of purified catalytic fragments of QDE-1 Ncr and QDE-1 Tte were assayed at 30–60 °C in the presence of an ssRNA ( A ) or an ssDNA template ( B ). The reaction products were separated by native agarose gel electrophoresis and visualized using ethidium bromide staining or 32 P phosphorimaging, as indicated. Note that QDE-1 Tte is substantially more efficient than QDE-1 Ncr in generating sRNA products that migrate either at a low molecular weight position or in a template base-paired form. C , RNA polymerase activities of QDE-1 Ncr and QDE-1 Tte were assayed at 45 °C in the presence of a ssRNA template and analyzed by denaturing agarose gel electrophoresis. D , RNA products from C were incubated with increasing concentrations of RNase ONE or RNase ONE reaction buffer, as specified under “Experimental Procedures.” Positions of the 1× full template-length products of processive end-to-end polymerization initiated in a primer-independent manner and sRNA products of non-processive polymerization are indicated on the right . Also shown is an expected position of “back-primed” 2× template-length products, which QDE-1 Ncr can generate for some but not all ssRNA templates ( 33 ).

Techniques Used: Purification, Agarose Gel Electrophoresis, Staining, Molecular Weight, Incubation

15) Product Images from "RIG-I ATPase Activity and Discrimination of Self-RNA versus Non-Self-RNA"

Article Title: RIG-I ATPase Activity and Discrimination of Self-RNA versus Non-Self-RNA

Journal: mBio

doi: 10.1128/mBio.02349-14

Ribonucleotides on the bottom strand are required for RIG-I ATPase activity. (A to G) Purified his-RIG-I (200 nM) was incubated with [γ- 32 P]ATP in the presence of increasing amounts (4 to 250 nM) of various RNA or RNA/DNA hybrids as indicated (only the data from the 250 nM concentration are shown here; the complete range is shown in Fig. S2 in the supplemental material). (A) Importance of the 5′ ppp. (B and G) Importance of a base-paired 5′ end. (C to F) Nature of the bottom strand. Reactions were revealed and quantified by phosphorimaging. ATPase data are presented as relative (Rel.) ATPase activities normalized to the ATPase activity of RIG-I in the absence of RNA. Data are represented as means ± standard errors of the means (SEM) ( n = 4) of the 250 nM RNA concentration. Significance: NS, P > 0.05; *, 0.01 ≤ P
Figure Legend Snippet: Ribonucleotides on the bottom strand are required for RIG-I ATPase activity. (A to G) Purified his-RIG-I (200 nM) was incubated with [γ- 32 P]ATP in the presence of increasing amounts (4 to 250 nM) of various RNA or RNA/DNA hybrids as indicated (only the data from the 250 nM concentration are shown here; the complete range is shown in Fig. S2 in the supplemental material). (A) Importance of the 5′ ppp. (B and G) Importance of a base-paired 5′ end. (C to F) Nature of the bottom strand. Reactions were revealed and quantified by phosphorimaging. ATPase data are presented as relative (Rel.) ATPase activities normalized to the ATPase activity of RIG-I in the absence of RNA. Data are represented as means ± standard errors of the means (SEM) ( n = 4) of the 250 nM RNA concentration. Significance: NS, P > 0.05; *, 0.01 ≤ P

Techniques Used: Activity Assay, Purification, Incubation, Concentration Assay

RIG-I oligomerization on RNA hybrids is RNA length dependent and ATPase independent. EMSA analysis was performed by incubating various radiolabeled RNA hybrids (250 nM) with purified his-RIG-I (2.5 μM (A and E); with increasing amount of RIG-I (4, 6, or 8 μM) in the presence of ATP and MgCl 2 (B to D)). (A) RIG-I binding and oligomerization are independent of the ATPase activity. (B) dsRNA length dependence for RIG-I oligomerization. (C) The longer the dsRNA hybrid, the more easily 2-RIG-I/RNA or 3-RIG-I/RNA complexes are formed. (D) RIG-I binds ssRNA. (E) Importance of ribonucleotide content. The 2*5*d-18r molecule forms a RIG-I dimer, in contrast to its mirror molecule, 2*5*r-18d. Reactions were analyzed on native gels and revealed by phosphorimaging. See also Fig. S4 in the supplemental material.
Figure Legend Snippet: RIG-I oligomerization on RNA hybrids is RNA length dependent and ATPase independent. EMSA analysis was performed by incubating various radiolabeled RNA hybrids (250 nM) with purified his-RIG-I (2.5 μM (A and E); with increasing amount of RIG-I (4, 6, or 8 μM) in the presence of ATP and MgCl 2 (B to D)). (A) RIG-I binding and oligomerization are independent of the ATPase activity. (B) dsRNA length dependence for RIG-I oligomerization. (C) The longer the dsRNA hybrid, the more easily 2-RIG-I/RNA or 3-RIG-I/RNA complexes are formed. (D) RIG-I binds ssRNA. (E) Importance of ribonucleotide content. The 2*5*d-18r molecule forms a RIG-I dimer, in contrast to its mirror molecule, 2*5*r-18d. Reactions were analyzed on native gels and revealed by phosphorimaging. See also Fig. S4 in the supplemental material.

Techniques Used: Purification, Binding Assay, Activity Assay

16) Product Images from "Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells"

Article Title: Polyadenylation and degradation of structurally abnormal mitochondrial tRNAs in human cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky159

Mitochondrial tRNA Ser(UCN) is modified in cells exposed to high levels of EtBr. ( A ) Northern blots probed for mitochondrial tRNA Ser(UCN) and cytosolic tRNA Ser(UCH) from wild-type and 7472insC mutant 143B cybrid cells treated with 2.5 μg/ml EtBr for the indicated times. For similar blots, where the positions of size markers were determined, see Supplementary Figure S2F . ( B, C ) Quantitation by phosphorimaging of Northern blots probed for mitochondrial tRNA Ser(UCN) during 24 h of EtBr treatment. Data were first adjusted for background, then normalized against 5S rRNA, and finally against the corresponding signals at t = 0: (B) levels of mature tRNA Ser(UCN) and proportion of signal corresponding to modified tRNA migrating at higher apparent molecular weight. For data from a second, independent experiment, as well as data averaged from the two experiments, see Supplementary Figure S1A . (C) Levels of total tRNA Ser(UCN) , in this case averages for the two experiments, showing very similar profiles for wild-type and mutant cybrid cells.
Figure Legend Snippet: Mitochondrial tRNA Ser(UCN) is modified in cells exposed to high levels of EtBr. ( A ) Northern blots probed for mitochondrial tRNA Ser(UCN) and cytosolic tRNA Ser(UCH) from wild-type and 7472insC mutant 143B cybrid cells treated with 2.5 μg/ml EtBr for the indicated times. For similar blots, where the positions of size markers were determined, see Supplementary Figure S2F . ( B, C ) Quantitation by phosphorimaging of Northern blots probed for mitochondrial tRNA Ser(UCN) during 24 h of EtBr treatment. Data were first adjusted for background, then normalized against 5S rRNA, and finally against the corresponding signals at t = 0: (B) levels of mature tRNA Ser(UCN) and proportion of signal corresponding to modified tRNA migrating at higher apparent molecular weight. For data from a second, independent experiment, as well as data averaged from the two experiments, see Supplementary Figure S1A . (C) Levels of total tRNA Ser(UCN) , in this case averages for the two experiments, showing very similar profiles for wild-type and mutant cybrid cells.

Techniques Used: Modification, Northern Blot, Mutagenesis, Quantitation Assay, Molecular Weight

Enzymes required for mitochondrial tRNA polyadenylation and turnover. Northern blots probed for mitochondrial tRNA Ser(UCN) , following EtBr treatment in 143B wild-type cybrid cells also treated with cordycepin (CDY) or siRNAs against different genes as indicated. ( A ) Cells pre-treated for 1 h and then continuously throughout the experiment with or without 20 μg/ml cordycepin, as shown. ( B ) Cells pre-treated for 96 h with siRNAs as indicated (see Supplementary Table S1 ), or mock-transfected, before 6 h of treatment with or without EtBr, as shown. ( C ) Cells pre-treated for 72 h with siRNAs as indicated (see Supplementary Table S1 ), or untreated, before 6 h of treatment with or without EtBr, followed by recovery for the indicated times after removal of the drug. In each siRNA experiment shown, knockdown at the protein level was verified by western blots, representative examples of which are shown in Supplementary Figure S4B and D . 72 h or 96 h of knockdown gave essentially identical results, both on Western and Northern blots. Signals detected by phosphorimaging in parts (B, C). Note that we confirmed the effects of knockdown of mtPAP, SUV3 and PNPase using alternative siRNAs ( Supplementary Figure S4E, F, G ).
Figure Legend Snippet: Enzymes required for mitochondrial tRNA polyadenylation and turnover. Northern blots probed for mitochondrial tRNA Ser(UCN) , following EtBr treatment in 143B wild-type cybrid cells also treated with cordycepin (CDY) or siRNAs against different genes as indicated. ( A ) Cells pre-treated for 1 h and then continuously throughout the experiment with or without 20 μg/ml cordycepin, as shown. ( B ) Cells pre-treated for 96 h with siRNAs as indicated (see Supplementary Table S1 ), or mock-transfected, before 6 h of treatment with or without EtBr, as shown. ( C ) Cells pre-treated for 72 h with siRNAs as indicated (see Supplementary Table S1 ), or untreated, before 6 h of treatment with or without EtBr, followed by recovery for the indicated times after removal of the drug. In each siRNA experiment shown, knockdown at the protein level was verified by western blots, representative examples of which are shown in Supplementary Figure S4B and D . 72 h or 96 h of knockdown gave essentially identical results, both on Western and Northern blots. Signals detected by phosphorimaging in parts (B, C). Note that we confirmed the effects of knockdown of mtPAP, SUV3 and PNPase using alternative siRNAs ( Supplementary Figure S4E, F, G ).

Techniques Used: Northern Blot, Transfection, Western Blot

Mitochondrial tRNAs are differentially modified in cells exposed to high levels of EtBr. ( A ) Northern blots probed for the indicated mitochondrial tRNAs in wild-type 143B cybrid cells treated with EtBr for the indicated times (tRNA Phe – 2.5 μg/ml EtBr, tRNA Leu(UUR) and tRNA Ser(AGY) – 3.5 μg/ml EtBr. ( B ) Quantitation by phosphorimaging of Northern blots probed for the indicated mitochondrial tRNAs in wild-type 143B cybrid cells treated with 2.5 μg/ml EtBr for the indicated times. Data for mature and modified tRNAs were first adjusted for background, then normalized against 5S rRNA, and finally against the corresponding signals at t = 0. In each case the amounts of mature tRNA, the proportion of modified tRNA and the total amount are shown. For data from a second, independent experiment, see Supplementary Figure S2A .
Figure Legend Snippet: Mitochondrial tRNAs are differentially modified in cells exposed to high levels of EtBr. ( A ) Northern blots probed for the indicated mitochondrial tRNAs in wild-type 143B cybrid cells treated with EtBr for the indicated times (tRNA Phe – 2.5 μg/ml EtBr, tRNA Leu(UUR) and tRNA Ser(AGY) – 3.5 μg/ml EtBr. ( B ) Quantitation by phosphorimaging of Northern blots probed for the indicated mitochondrial tRNAs in wild-type 143B cybrid cells treated with 2.5 μg/ml EtBr for the indicated times. Data for mature and modified tRNAs were first adjusted for background, then normalized against 5S rRNA, and finally against the corresponding signals at t = 0. In each case the amounts of mature tRNA, the proportion of modified tRNA and the total amount are shown. For data from a second, independent experiment, see Supplementary Figure S2A .

Techniques Used: Modification, Northern Blot, Quantitation Assay

17) Product Images from "Sinorhizobium meliloti SyrA Mediates the Transcriptional Regulation of Genes Involved in Lipopolysaccharide Sulfation and Exopolysaccharide Biosynthesis ▿"

Article Title: Sinorhizobium meliloti SyrA Mediates the Transcriptional Regulation of Genes Involved in Lipopolysaccharide Sulfation and Exopolysaccharide Biosynthesis ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.01803-06

Increased LpsS activity in strains overexpressing SyrA. Plasmids harboring S. meliloti host-specific nod genes were introduced into Rm1021 (wild type). In addition, the exoR ::Tn 5 and exoS ::Tn 5 regulatory mutations were introduced into Rm1021 by transduction. (A) LPS sulfotransferase activity. The strains were grown to saturation (OD 600 of 2.5) and extracts were assayed for LPS sulfotransferase activity as described in Materials and Methods. LPS sulfotransferase activity represents sulfate incorporated into LPS (as measured by phosphorimaging)/mg of protein. Lane 1, Rm1021 (wild type)/pTE3 (vector); lane 2, Rm1021/pRmJT5 (which contains a 20-kb fragment of pSymA with host-specific nod genes); lane 3 Rm1021/pTE3:: syrM ; lane 4, Rm1021/pTE3:: syrA ; lane 5, exoR ::Tn 5 ; lane 6, exoS ::Tn 5 ; lane 7, chvI ( K214T )/pTE3; lane 8, chvI ( K214T )/pTE3:: syrA ; lane 9, lpsS ::pDW33/pTE3; lane 10, lpsS ::pDW33/pTE3:: syrA . Error bars represent standard deviations of experiments carried out in triplicate. (B) LPS sulfation in strains overexpressing SyrA. Strains were grown to saturation (OD 600 of 2.5) in the presence of Na 2 35 SO 4 (ICN). Cell surface polysaccharides were then extracted and fractionated by Tris-Tricine-PAGE, and the incorporation of sulfate was measured by phosphorimaging as described in Materials and Methods. Lane 1, Rm1021 (wild type)/pTE3 (vector); lane 2, Rm1021/pRmJT5 (which contains a 20-kb fragment of pSymA with host-specific nod genes); lane 3, Rm1021/pTE3:: syrM ; lane 4, Rm1021/pTE3:: syrA ; lane 5, exoR ::Tn 5 ; lane 6, exoS ::Tn 5 . (C) Measurement of PAPS biosynthesis. Strains were cultured in the presence of 35 SO 4 , and PAPS and APS (adenosine-5′-phosphosulfate, a derivative of PAPS) was recovered by formic acid extraction as described in Materials and Methods. The formic acid extracts were then subjected to fractionation on PEI-cellulose, and the radioactive material was detected by phosphorimaging. Migration of PAPS and APS was determined by comparison to labeled standards (not shown). The asterisk signifies a high mobility spot that did not comigrate with any of the standards. Lane 1, Rm1021 (wild type) containing pTE3 (Vect) and pMS03 (Vect); lane 2, Rm1021 containing pTE3:: syrA and pMS03; lane 3, Rm1021 containing pTE3 and pMS03:: nodPQ ; lane 4, Rm1021 containing pTE3:: syrA and pMS03:: nodPQ . (D) Overexpression of nodPQ results in a SyrA-dependent increase in LPS sulfation. Either pMS03 (vector control) or pMS03 containing nodPQ from M. loti was introduced into wild-type strains harboring either pTE3 or pTE3:: syrA . The incorporation of sulfate was then measured as described in panel B. Lane 1, Rm1021 (wild type), containing pTE3 (Vect) and pMS03 (Vect); lane 2, Rm1021 containing pTE3:: syrA and pMS03; lane 3, Rm1021 containing pTE3 and pMS03:: nodPQ ; lane 4, Rm1021 containing pTE3:: syrA and pMS03:: nodPQ ; lane 5, exoS ::Tn 5 containing pMS03; lane 6, exoS ::Tn 5 containing pMS03:: nodPQ ; lane 7, lpsS ::pDW33 containing pTE3 and pMS03; lane 8, lpsS ::pDW33 containing pTE3:: syrA and pMS03; lane 9, lpsS ::pDW33 containing pTE3 and pMS03:: nodPQ ; lane 10, lpsS ::pDW33 containing pTE3:: syrA and pMS03:: nodPQ. Error bars represent standard deviations of experiments carried out in triplicate.
Figure Legend Snippet: Increased LpsS activity in strains overexpressing SyrA. Plasmids harboring S. meliloti host-specific nod genes were introduced into Rm1021 (wild type). In addition, the exoR ::Tn 5 and exoS ::Tn 5 regulatory mutations were introduced into Rm1021 by transduction. (A) LPS sulfotransferase activity. The strains were grown to saturation (OD 600 of 2.5) and extracts were assayed for LPS sulfotransferase activity as described in Materials and Methods. LPS sulfotransferase activity represents sulfate incorporated into LPS (as measured by phosphorimaging)/mg of protein. Lane 1, Rm1021 (wild type)/pTE3 (vector); lane 2, Rm1021/pRmJT5 (which contains a 20-kb fragment of pSymA with host-specific nod genes); lane 3 Rm1021/pTE3:: syrM ; lane 4, Rm1021/pTE3:: syrA ; lane 5, exoR ::Tn 5 ; lane 6, exoS ::Tn 5 ; lane 7, chvI ( K214T )/pTE3; lane 8, chvI ( K214T )/pTE3:: syrA ; lane 9, lpsS ::pDW33/pTE3; lane 10, lpsS ::pDW33/pTE3:: syrA . Error bars represent standard deviations of experiments carried out in triplicate. (B) LPS sulfation in strains overexpressing SyrA. Strains were grown to saturation (OD 600 of 2.5) in the presence of Na 2 35 SO 4 (ICN). Cell surface polysaccharides were then extracted and fractionated by Tris-Tricine-PAGE, and the incorporation of sulfate was measured by phosphorimaging as described in Materials and Methods. Lane 1, Rm1021 (wild type)/pTE3 (vector); lane 2, Rm1021/pRmJT5 (which contains a 20-kb fragment of pSymA with host-specific nod genes); lane 3, Rm1021/pTE3:: syrM ; lane 4, Rm1021/pTE3:: syrA ; lane 5, exoR ::Tn 5 ; lane 6, exoS ::Tn 5 . (C) Measurement of PAPS biosynthesis. Strains were cultured in the presence of 35 SO 4 , and PAPS and APS (adenosine-5′-phosphosulfate, a derivative of PAPS) was recovered by formic acid extraction as described in Materials and Methods. The formic acid extracts were then subjected to fractionation on PEI-cellulose, and the radioactive material was detected by phosphorimaging. Migration of PAPS and APS was determined by comparison to labeled standards (not shown). The asterisk signifies a high mobility spot that did not comigrate with any of the standards. Lane 1, Rm1021 (wild type) containing pTE3 (Vect) and pMS03 (Vect); lane 2, Rm1021 containing pTE3:: syrA and pMS03; lane 3, Rm1021 containing pTE3 and pMS03:: nodPQ ; lane 4, Rm1021 containing pTE3:: syrA and pMS03:: nodPQ . (D) Overexpression of nodPQ results in a SyrA-dependent increase in LPS sulfation. Either pMS03 (vector control) or pMS03 containing nodPQ from M. loti was introduced into wild-type strains harboring either pTE3 or pTE3:: syrA . The incorporation of sulfate was then measured as described in panel B. Lane 1, Rm1021 (wild type), containing pTE3 (Vect) and pMS03 (Vect); lane 2, Rm1021 containing pTE3:: syrA and pMS03; lane 3, Rm1021 containing pTE3 and pMS03:: nodPQ ; lane 4, Rm1021 containing pTE3:: syrA and pMS03:: nodPQ ; lane 5, exoS ::Tn 5 containing pMS03; lane 6, exoS ::Tn 5 containing pMS03:: nodPQ ; lane 7, lpsS ::pDW33 containing pTE3 and pMS03; lane 8, lpsS ::pDW33 containing pTE3:: syrA and pMS03; lane 9, lpsS ::pDW33 containing pTE3 and pMS03:: nodPQ ; lane 10, lpsS ::pDW33 containing pTE3:: syrA and pMS03:: nodPQ. Error bars represent standard deviations of experiments carried out in triplicate.

Techniques Used: Activity Assay, Transduction, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Papanicolaou Stain, Cell Culture, Fractionation, Migration, Labeling, Over Expression

18) Product Images from "Functional Evolution in Orthologous Cell-encoded RNA-dependent RNA Polymerases *"

Article Title: Functional Evolution in Orthologous Cell-encoded RNA-dependent RNA Polymerases *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.685933

QDE-1 orthologs have distinct enzymatic properties. A and B , RNA polymerase activities of purified catalytic fragments of QDE-1 Ncr and QDE-1 Tte were assayed at 30–60 °C in the presence of an ssRNA ( A ) or an ssDNA template ( B ). The reaction products were separated by native agarose gel electrophoresis and visualized using ethidium bromide staining or 32 P phosphorimaging, as indicated. Note that QDE-1 Tte is substantially more efficient than QDE-1 Ncr in generating sRNA products that migrate either at a low molecular weight position or in a template base-paired form. C , RNA polymerase activities of QDE-1 Ncr and QDE-1 Tte were assayed at 45 °C in the presence of a ssRNA template and analyzed by denaturing agarose gel electrophoresis. D , RNA products from C were incubated with increasing concentrations of RNase ONE or RNase ONE reaction buffer, as specified under “Experimental Procedures.” Positions of the 1× full template-length products of processive end-to-end polymerization initiated in a primer-independent manner and sRNA products of non-processive polymerization are indicated on the right . Also shown is an expected position of “back-primed” 2× template-length products, which QDE-1 Ncr can generate for some but not all ssRNA templates ( 33 ).
Figure Legend Snippet: QDE-1 orthologs have distinct enzymatic properties. A and B , RNA polymerase activities of purified catalytic fragments of QDE-1 Ncr and QDE-1 Tte were assayed at 30–60 °C in the presence of an ssRNA ( A ) or an ssDNA template ( B ). The reaction products were separated by native agarose gel electrophoresis and visualized using ethidium bromide staining or 32 P phosphorimaging, as indicated. Note that QDE-1 Tte is substantially more efficient than QDE-1 Ncr in generating sRNA products that migrate either at a low molecular weight position or in a template base-paired form. C , RNA polymerase activities of QDE-1 Ncr and QDE-1 Tte were assayed at 45 °C in the presence of a ssRNA template and analyzed by denaturing agarose gel electrophoresis. D , RNA products from C were incubated with increasing concentrations of RNase ONE or RNase ONE reaction buffer, as specified under “Experimental Procedures.” Positions of the 1× full template-length products of processive end-to-end polymerization initiated in a primer-independent manner and sRNA products of non-processive polymerization are indicated on the right . Also shown is an expected position of “back-primed” 2× template-length products, which QDE-1 Ncr can generate for some but not all ssRNA templates ( 33 ).

Techniques Used: Purification, Agarose Gel Electrophoresis, Staining, Molecular Weight, Incubation

19) Product Images from "Rex1p deficiency leads to accumulation of precursor initiator tRNAMet and polyadenylation of substrate RNAs in Saccharomyces cerevisiae"

Article Title: Rex1p deficiency leads to accumulation of precursor initiator tRNAMet and polyadenylation of substrate RNAs in Saccharomyces cerevisiae

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkn925

Loss of Rex1p results in polyadenylation of 5S rRNA and tRNA Arg from the dimeric tRNA Arg -tRNA Asp transcript. ( A ) Total and poly (A) + RNA were analyzed as described for Figure 4 . The blot was probed for 5S rRNA (JA99) and the results visualized by autoradiography. ( B ) Northern analysis was performed using a labeled oligonucleotide (JA557) that hybridizes to the 3′ end of tRNA Arg and the linker sequence between the two tRNAs. Phosphorimaging was used to analyze results. The presence of mature forms of RNAs in the poly(A) + lanes represent RNAs not eliminated during oligo-(dT) selection.
Figure Legend Snippet: Loss of Rex1p results in polyadenylation of 5S rRNA and tRNA Arg from the dimeric tRNA Arg -tRNA Asp transcript. ( A ) Total and poly (A) + RNA were analyzed as described for Figure 4 . The blot was probed for 5S rRNA (JA99) and the results visualized by autoradiography. ( B ) Northern analysis was performed using a labeled oligonucleotide (JA557) that hybridizes to the 3′ end of tRNA Arg and the linker sequence between the two tRNAs. Phosphorimaging was used to analyze results. The presence of mature forms of RNAs in the poly(A) + lanes represent RNAs not eliminated during oligo-(dT) selection.

Techniques Used: Autoradiography, Northern Blot, Labeling, Sequencing, Selection

Changes in the 3′ trailer length influence tRNA i Met accumulation in a trm6-504 rex1Δ strain. ( A ) Wt (Y200) or trm6-504 (Y190) strains carrying vector (YCplac33), hc IMT4 , or hc imt4-3 were grown at 30°C for 2 days on SC −URA . ( B ) Total RNA (20 μg) isolated from Wt (Y200), trm6-504 (Y190), trm6-504 rex1Δ (Y386) and rex1Δ (Y387) strains carrying empty vector (YCplac33), hc IMT4 (pJA108) or hc imt4-3 (p467) was subjected to Northern analysis. The blot was probed with a radiolabeled oligonucleotide complementary to the 5′ leader of IMT4 (JA66), and the results visualized using phosphorimaging. The blot was stripped, probed again with an oligonucleotide that hybridizes to 5S rRNA (JA99), and analyzed by phosphorimaging.
Figure Legend Snippet: Changes in the 3′ trailer length influence tRNA i Met accumulation in a trm6-504 rex1Δ strain. ( A ) Wt (Y200) or trm6-504 (Y190) strains carrying vector (YCplac33), hc IMT4 , or hc imt4-3 were grown at 30°C for 2 days on SC −URA . ( B ) Total RNA (20 μg) isolated from Wt (Y200), trm6-504 (Y190), trm6-504 rex1Δ (Y386) and rex1Δ (Y387) strains carrying empty vector (YCplac33), hc IMT4 (pJA108) or hc imt4-3 (p467) was subjected to Northern analysis. The blot was probed with a radiolabeled oligonucleotide complementary to the 5′ leader of IMT4 (JA66), and the results visualized using phosphorimaging. The blot was stripped, probed again with an oligonucleotide that hybridizes to 5S rRNA (JA99), and analyzed by phosphorimaging.

Techniques Used: Plasmid Preparation, Isolation, Northern Blot

Rex1p displays tRNA 3′end processing activity in vivo and in vitro . ( A ) A diagram of Rex1p showing the conserved amino acids found in the three Exo motifs. An alignment of Rex1, 2, 3 and 4, and E. coli RNase T protein sequence is shown underneath to illustrate the conservation of the D × E sequence found in the ExoI domain. ( B ) Wt and mutant FLAG-tagged Rex1p were purified from yeast (strains Y438 and Y495, respectively) using affinity chromatography. Purified protein was subjected to SDS-PAGE and visualized by Coomassie staining. The positions of molecular weight standards (Broad Range Protein Marker, New England Bioloabs) are indicated. ( C ) Gel purified 32 P- 5′ end labeled tRNAs i Met (∼ 10 pM) were incubated in buffer alone or with Wt or mutant Rex1p (∼7.5 nM) at 30°C for 10 min. Reaction products were separated on a 10% denaturing polyacrylamide gel and visualized by autoradiography. The positions of RNAs of known length (Decade Marker, Ambion) are indicated (in nucleotides). ( D ) Wt Rex1p (∼7.5 nM) was incubated with labeled ‘ IMT3 ’ tRNA i Met (∼20 pM) for known periods of time from 0.5–7 min, as indicated. Control reactions with ‘ IMT3 ’ tRNA i Met lacking either enzyme or Mg 2+ are shown, as well as a control reaction lacking enzyme that contained the ‘mature’ tRNA i Met . After separation on a denaturing 10% polyacrylamide gel, results were visualized using phosphorimaging. ( E ) Northern analysis of total RNA (10 μg) isolated from a trm6-504 rex1Δ strain carrying an empty vector (pRS316), or a plasmid with galactose-inducible Wt REX1 (pAV101) or mutant rex1 (p532), grown under non-inducing or inducing conditions. The blot was probed with a radiolabeled oligonucleotide complementary to tRNA i Met (JA11) and the results visualized using autoradiography.
Figure Legend Snippet: Rex1p displays tRNA 3′end processing activity in vivo and in vitro . ( A ) A diagram of Rex1p showing the conserved amino acids found in the three Exo motifs. An alignment of Rex1, 2, 3 and 4, and E. coli RNase T protein sequence is shown underneath to illustrate the conservation of the D × E sequence found in the ExoI domain. ( B ) Wt and mutant FLAG-tagged Rex1p were purified from yeast (strains Y438 and Y495, respectively) using affinity chromatography. Purified protein was subjected to SDS-PAGE and visualized by Coomassie staining. The positions of molecular weight standards (Broad Range Protein Marker, New England Bioloabs) are indicated. ( C ) Gel purified 32 P- 5′ end labeled tRNAs i Met (∼ 10 pM) were incubated in buffer alone or with Wt or mutant Rex1p (∼7.5 nM) at 30°C for 10 min. Reaction products were separated on a 10% denaturing polyacrylamide gel and visualized by autoradiography. The positions of RNAs of known length (Decade Marker, Ambion) are indicated (in nucleotides). ( D ) Wt Rex1p (∼7.5 nM) was incubated with labeled ‘ IMT3 ’ tRNA i Met (∼20 pM) for known periods of time from 0.5–7 min, as indicated. Control reactions with ‘ IMT3 ’ tRNA i Met lacking either enzyme or Mg 2+ are shown, as well as a control reaction lacking enzyme that contained the ‘mature’ tRNA i Met . After separation on a denaturing 10% polyacrylamide gel, results were visualized using phosphorimaging. ( E ) Northern analysis of total RNA (10 μg) isolated from a trm6-504 rex1Δ strain carrying an empty vector (pRS316), or a plasmid with galactose-inducible Wt REX1 (pAV101) or mutant rex1 (p532), grown under non-inducing or inducing conditions. The blot was probed with a radiolabeled oligonucleotide complementary to tRNA i Met (JA11) and the results visualized using autoradiography.

Techniques Used: Activity Assay, In Vivo, In Vitro, Sequencing, Mutagenesis, Purification, Affinity Chromatography, SDS Page, Staining, Molecular Weight, Marker, Labeling, Incubation, Autoradiography, Northern Blot, Isolation, Plasmid Preparation

20) Product Images from "Programmable RNA recognition and cleavage by CRISPR/Cas9"

Article Title: Programmable RNA recognition and cleavage by CRISPR/Cas9

Journal: Nature

doi: 10.1038/nature13769

RNA cleavage is marginally stimulated by di- and trideoxyribonucleotide PAMmers Cleavage reactions contained ~1 nM 5′-radiolabelled target ssRNA and no PAMmer (left), 100 nM 18-nt PAMmer (second from left), or 1 mM of the indicated di- or tri-nucleotide (remaining lanes). Reaction products were resolved by 12% denaturing polyacrylamide gel electrophoresis (PAGE) and visualized by phosphorimaging.
Figure Legend Snippet: RNA cleavage is marginally stimulated by di- and trideoxyribonucleotide PAMmers Cleavage reactions contained ~1 nM 5′-radiolabelled target ssRNA and no PAMmer (left), 100 nM 18-nt PAMmer (second from left), or 1 mM of the indicated di- or tri-nucleotide (remaining lanes). Reaction products were resolved by 12% denaturing polyacrylamide gel electrophoresis (PAGE) and visualized by phosphorimaging.

Techniques Used: Polyacrylamide Gel Electrophoresis

21) Product Images from "The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu"

Article Title: The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu

Journal: Nature chemical biology

doi: 10.1038/nchembio.1364

Doc phosphorylates EF-Tu at the conserved Thr382 (a). Radiolabeling of EF-Tu by Doc added during (blue) or after (for 15 min; red) ternary complex formation in the presence of γ[ 32 P]-GTP (here and after 3 pmol unless otherwise specified) was analyzed by SDS-PAGE and autoradiography. (b). Radiolabeling of EF-Tu by Doc in the presence of α[ 32 P]-ATP, γ[ 32 P]-ATP, α[ 32 P]-GTP or γ[ 32 P]-GTP. (c). Products of EF-Tu modification (30 s) by Doc in the presence of α[ 32 P]-ATP or γ[ 32 P]-ATP were separated by thin layer chromatography (PEI-cellulose in 0.5M K 2 HPO 4 ) and analyzed by autoradiography. ATP and ADP mobility standards were visualized under UV254 35 , 36 and marked with radioactive spots before phosphorimaging. (d). Phosphorylation of EF-Tu by catalytic mutant of Doc, H66Y. (e). Dipeptide MF synthesis with EF-Tu and EF-Tu T382V in the absence or presence of Doc added before ternary complex formation. (f). Phosphorylation of wild-type EF-Tu and mutant EF-Tu T382V . (g). Doc and γ[ 32 P]-ATP were added to purified EF-Tu, S30 or S100 lysate fractions. A band migrating above the purified EF-Tu corresponds to in vitro aggregated EF-Tu 37 .
Figure Legend Snippet: Doc phosphorylates EF-Tu at the conserved Thr382 (a). Radiolabeling of EF-Tu by Doc added during (blue) or after (for 15 min; red) ternary complex formation in the presence of γ[ 32 P]-GTP (here and after 3 pmol unless otherwise specified) was analyzed by SDS-PAGE and autoradiography. (b). Radiolabeling of EF-Tu by Doc in the presence of α[ 32 P]-ATP, γ[ 32 P]-ATP, α[ 32 P]-GTP or γ[ 32 P]-GTP. (c). Products of EF-Tu modification (30 s) by Doc in the presence of α[ 32 P]-ATP or γ[ 32 P]-ATP were separated by thin layer chromatography (PEI-cellulose in 0.5M K 2 HPO 4 ) and analyzed by autoradiography. ATP and ADP mobility standards were visualized under UV254 35 , 36 and marked with radioactive spots before phosphorimaging. (d). Phosphorylation of EF-Tu by catalytic mutant of Doc, H66Y. (e). Dipeptide MF synthesis with EF-Tu and EF-Tu T382V in the absence or presence of Doc added before ternary complex formation. (f). Phosphorylation of wild-type EF-Tu and mutant EF-Tu T382V . (g). Doc and γ[ 32 P]-ATP were added to purified EF-Tu, S30 or S100 lysate fractions. A band migrating above the purified EF-Tu corresponds to in vitro aggregated EF-Tu 37 .

Techniques Used: Radioactivity, SDS Page, Autoradiography, Modification, Thin Layer Chromatography, Mutagenesis, Purification, In Vitro

Doc inhibits translation by inactivation of ternary complex formation (a). Ribbon representation of the structural superposition of Doc (pdbid 3K33 25 ) onto Fic adenylylase from N. meningitides (NmFic; pdbid 3S6A 1 ). Doc is colored in blue and NmFic in pink, with respective catalytic loops in light and dark green. (b). Superposition of the catalytic loops of Doc (residues H66-R74) and NmFic (in the nucleotide bound conformation; residues H107-R114). The AMPPNP molecule bound to NmFic is shown in black (c) . Luciferase synthesis in a commercially available cell-free translation system was performed in the presence of [ 35 S]-methionine in the absence or presence of Doc and revealed by phosphorimaging. Here and after, full images of gels, TLCs and TLEs are presented in Supplementary Fig. 1 . (d) . Scheme of the assembly of an in vitro translation system using purified components (PK-pyruvate kinase; PEP-phosphoenol pyruvate). Steps at which Doc was added to the reactions in panels (e) and (f) and in Figure 2a , e are depicted in colors. (e) Synthesis of dipeptide MF in the absence or presence of Doc added after (red) or during (blue) ternary complex (TC) formation. (f). Synthesis of tripeptide MFV in the presence or absence of Doc added during initiation (cyan) or ternary (blue) complex formation. Peptides were analyzed by thin layer electrophoresis and autoradiography 14 .
Figure Legend Snippet: Doc inhibits translation by inactivation of ternary complex formation (a). Ribbon representation of the structural superposition of Doc (pdbid 3K33 25 ) onto Fic adenylylase from N. meningitides (NmFic; pdbid 3S6A 1 ). Doc is colored in blue and NmFic in pink, with respective catalytic loops in light and dark green. (b). Superposition of the catalytic loops of Doc (residues H66-R74) and NmFic (in the nucleotide bound conformation; residues H107-R114). The AMPPNP molecule bound to NmFic is shown in black (c) . Luciferase synthesis in a commercially available cell-free translation system was performed in the presence of [ 35 S]-methionine in the absence or presence of Doc and revealed by phosphorimaging. Here and after, full images of gels, TLCs and TLEs are presented in Supplementary Fig. 1 . (d) . Scheme of the assembly of an in vitro translation system using purified components (PK-pyruvate kinase; PEP-phosphoenol pyruvate). Steps at which Doc was added to the reactions in panels (e) and (f) and in Figure 2a , e are depicted in colors. (e) Synthesis of dipeptide MF in the absence or presence of Doc added after (red) or during (blue) ternary complex (TC) formation. (f). Synthesis of tripeptide MFV in the presence or absence of Doc added during initiation (cyan) or ternary (blue) complex formation. Peptides were analyzed by thin layer electrophoresis and autoradiography 14 .

Techniques Used: Luciferase, In Vitro, Purification, Electrophoresis, Autoradiography

22) Product Images from "Phase transitions in the assembly and function of human miRISC"

Article Title: Phase transitions in the assembly and function of human miRISC

Journal: Cell

doi: 10.1016/j.cell.2018.02.051

Biochemical characterization of Ago2-TNRC6B droplets ( A ) Ago2-TNRC6B droplets can be separated from the bulk solvent by centrifugation. Cartoon schematic of procedure (left), and images of droplets in input and supernatant fractions (right). ( B ) Droplets recruit full-length TNRC6B, Ago2, and miRNA target RNAs. TNRC6B (~1 mM, partially purified) was mixed with Ago2 (0.5 µM) loaded with either let-7 or miR122, and a 32 P-labeled let-7 target RNA (8xlet7 target, ~3 nM). After centrifugation, supernatant and pellet fractions were analyzed by Coomassie stained SDS PAGE (right, top panel) and phosphorimaging of a denaturing gel (right, bottom panel). ( C ) Ago2 remains active in the separated phase. TNRC6B (~ 1 µM) was mixed with Ago2-miR122 (250 nM) and a 32 P-labeled target RNA (~0.5 µM) with perfect complementarity to miR122 in the absence of divalent cations. After centrifugation MgCl 2 (3 mM) was added to the separated phase. Target RNA was extracted and analyzed by denaturing PAGE and phosphorimaging (right panel). ( D ) TNRC6B-Ago2 droplets recruit other miRISC components. TNRC6B (40 nM) was mixed with Ago2 (40 nM) and soluble lysate from HEK 293 cells (OD 260 .
Figure Legend Snippet: Biochemical characterization of Ago2-TNRC6B droplets ( A ) Ago2-TNRC6B droplets can be separated from the bulk solvent by centrifugation. Cartoon schematic of procedure (left), and images of droplets in input and supernatant fractions (right). ( B ) Droplets recruit full-length TNRC6B, Ago2, and miRNA target RNAs. TNRC6B (~1 mM, partially purified) was mixed with Ago2 (0.5 µM) loaded with either let-7 or miR122, and a 32 P-labeled let-7 target RNA (8xlet7 target, ~3 nM). After centrifugation, supernatant and pellet fractions were analyzed by Coomassie stained SDS PAGE (right, top panel) and phosphorimaging of a denaturing gel (right, bottom panel). ( C ) Ago2 remains active in the separated phase. TNRC6B (~ 1 µM) was mixed with Ago2-miR122 (250 nM) and a 32 P-labeled target RNA (~0.5 µM) with perfect complementarity to miR122 in the absence of divalent cations. After centrifugation MgCl 2 (3 mM) was added to the separated phase. Target RNA was extracted and analyzed by denaturing PAGE and phosphorimaging (right panel). ( D ) TNRC6B-Ago2 droplets recruit other miRISC components. TNRC6B (40 nM) was mixed with Ago2 (40 nM) and soluble lysate from HEK 293 cells (OD 260 .

Techniques Used: Centrifugation, Purification, Labeling, Staining, SDS Page, Polyacrylamide Gel Electrophoresis

Efficient target deadenylation in miRISC droplets ( A ) Schematic of experiment. ( B ) Deadenylation of a target RNA by miRISC. Ago2 (40 nM, final concentration), loaded with either let7 or miR122 (negative control), was mixed with a 32 P-5'-cap-labeled target RNA harboring binding sites for let-7 and a 114 nt. poly(A) tail in the presence of soluble lysate from HEK 293 cells (OD 260 ~3), with and without additional TNRC6B (~300 nM, partially purified). After a 15-minute incubation, supernatant and pellet fractions were isolated by centrifugation and target RNA was extracted and analyzed by denaturing PAGE and phosphorimaging. ( C ) Deadenylation timecourse. Reactions containing Ago2-let7 (40 nM) and soluble HEK 293 lysate (OD 260 ~3), with and without exogenous TNRC6B (~300 nM, final concentration) were fractionated at various times, and analyzed by denaturing gel. ( D ) Estimation of deadenylation rates. Target RNA bands in (C) were quantified and fraction of total intact RNA (A 114 ) for +/− TNRC6B conditions was plotted as a function of incubation time. Data were fit with a first order decay, yielding A 114 .
Figure Legend Snippet: Efficient target deadenylation in miRISC droplets ( A ) Schematic of experiment. ( B ) Deadenylation of a target RNA by miRISC. Ago2 (40 nM, final concentration), loaded with either let7 or miR122 (negative control), was mixed with a 32 P-5'-cap-labeled target RNA harboring binding sites for let-7 and a 114 nt. poly(A) tail in the presence of soluble lysate from HEK 293 cells (OD 260 ~3), with and without additional TNRC6B (~300 nM, partially purified). After a 15-minute incubation, supernatant and pellet fractions were isolated by centrifugation and target RNA was extracted and analyzed by denaturing PAGE and phosphorimaging. ( C ) Deadenylation timecourse. Reactions containing Ago2-let7 (40 nM) and soluble HEK 293 lysate (OD 260 ~3), with and without exogenous TNRC6B (~300 nM, final concentration) were fractionated at various times, and analyzed by denaturing gel. ( D ) Estimation of deadenylation rates. Target RNA bands in (C) were quantified and fraction of total intact RNA (A 114 ) for +/− TNRC6B conditions was plotted as a function of incubation time. Data were fit with a first order decay, yielding A 114 .

Techniques Used: Concentration Assay, Negative Control, Labeling, Binding Assay, Purification, Incubation, Isolation, Centrifugation, Polyacrylamide Gel Electrophoresis

23) Product Images from "Stage-Specific COPII-Mediated Cargo Selectivity in African Trypanosomes"

Article Title: Stage-Specific COPII-Mediated Cargo Selectivity in African Trypanosomes

Journal: mSphere

doi: 10.1128/msphere.00188-22

Transport of TbCatL in TbSec23/24 knockdowns. Specific dsRNA synthesis was induced for 48 h in TbSec23.2 (A), TbSec24.1 (B), TbSec23.1 (C), and TbSec24.2 (D) RNAi cell lines. Pulse (10 min)/Chase (2 h) radiolabeling was performed. TbCatL was immunoprecipitated from cell lysates at the indicated chase times and analyzed by SDS-PAGE and phosphorimaging (10 7 cells/lane). (Top) Phosphorimages of representative matched gels from control (Tet − ; upper) and silenced (Tet + ; lower). Mobilities of initial precursors (I and X) and the lysosomal mature (M) form are indicated. Matched Tet − and Tet + gels are from the same processed phosphorimage. (Bottom) Quantification of loss of the initial precursors (I and X). The nonspecific band (*) was not included in the quantification. Three biological replicates were quantified, and the data are presented as mean ± SD.
Figure Legend Snippet: Transport of TbCatL in TbSec23/24 knockdowns. Specific dsRNA synthesis was induced for 48 h in TbSec23.2 (A), TbSec24.1 (B), TbSec23.1 (C), and TbSec24.2 (D) RNAi cell lines. Pulse (10 min)/Chase (2 h) radiolabeling was performed. TbCatL was immunoprecipitated from cell lysates at the indicated chase times and analyzed by SDS-PAGE and phosphorimaging (10 7 cells/lane). (Top) Phosphorimages of representative matched gels from control (Tet − ; upper) and silenced (Tet + ; lower). Mobilities of initial precursors (I and X) and the lysosomal mature (M) form are indicated. Matched Tet − and Tet + gels are from the same processed phosphorimage. (Bottom) Quantification of loss of the initial precursors (I and X). The nonspecific band (*) was not included in the quantification. Three biological replicates were quantified, and the data are presented as mean ± SD.

Techniques Used: Radioactivity, Immunoprecipitation, SDS Page

p67 transport in TbSec23/24 knockdowns. Specific dsRNA synthesis was induced for 48 h in TbSec23.2 (A), TbSec24.1 (B), TbSec23.1 (C), and TbSec24.2 (D) RNAi cell lines. Pulse (15 min)/Chase (8 h) radiolabeling was performed. p67 was immunoprecipitated from cell lysates at the indicated chase times and analyzed by SDS-PAGE and phosphorimaging (10 7 cells/lane). (Top) Phosphorimages of representative matched gels from control (Tet − ) and silenced (Tet + ). Mobility of the gp100 precursor is indicated. Matched Tet − and Tet + gels are from the same processed phosphorimage. (Bottom) Quantification of loss of the initial ER precursor gp100. Three biological replicates were quantified, and the data are presented as mean ± SD.
Figure Legend Snippet: p67 transport in TbSec23/24 knockdowns. Specific dsRNA synthesis was induced for 48 h in TbSec23.2 (A), TbSec24.1 (B), TbSec23.1 (C), and TbSec24.2 (D) RNAi cell lines. Pulse (15 min)/Chase (8 h) radiolabeling was performed. p67 was immunoprecipitated from cell lysates at the indicated chase times and analyzed by SDS-PAGE and phosphorimaging (10 7 cells/lane). (Top) Phosphorimages of representative matched gels from control (Tet − ) and silenced (Tet + ). Mobility of the gp100 precursor is indicated. Matched Tet − and Tet + gels are from the same processed phosphorimage. (Bottom) Quantification of loss of the initial ER precursor gp100. Three biological replicates were quantified, and the data are presented as mean ± SD.

Techniques Used: Radioactivity, Immunoprecipitation, SDS Page

VSG transport in TbSec23/24 knockdowns. Specific dsRNA synthesis was induced for 48 h in TbSec23.2 (A), TbSec24.1 (B), TbSec23.1 (C), and TbSec24.2 (D) RNAi cell lines expressing VSG117. Pulse (15 min)/chase (4 h) radiolabeling was performed. CHX was added to block VSG synthesis, during the chase period. VSG was immunoprecipitated from cell lysates and media fractions at the indicated chase times and analyzed by SDS-PAGE and phosphorimaging (10 7 cells/lane). (Top) Phosphorimages of representative matched gels from control (Tet − ) and silenced (Tet + ) cell lines. Mobilities of full-length (V) and truncated (T) VSG are indicated. Vertical white spaces indicate lanes that were excised post-image processing for the sake of presentation. Matched Tet − and Tet + gels are from the same processed phosphorimage. (Bottom) Quantification of loss of initial full-length VSG from cells with concomitant release of truncated VSG to the media. Three biological replicates are quantified, and the data are presented as mean ± SD.
Figure Legend Snippet: VSG transport in TbSec23/24 knockdowns. Specific dsRNA synthesis was induced for 48 h in TbSec23.2 (A), TbSec24.1 (B), TbSec23.1 (C), and TbSec24.2 (D) RNAi cell lines expressing VSG117. Pulse (15 min)/chase (4 h) radiolabeling was performed. CHX was added to block VSG synthesis, during the chase period. VSG was immunoprecipitated from cell lysates and media fractions at the indicated chase times and analyzed by SDS-PAGE and phosphorimaging (10 7 cells/lane). (Top) Phosphorimages of representative matched gels from control (Tet − ) and silenced (Tet + ) cell lines. Mobilities of full-length (V) and truncated (T) VSG are indicated. Vertical white spaces indicate lanes that were excised post-image processing for the sake of presentation. Matched Tet − and Tet + gels are from the same processed phosphorimage. (Bottom) Quantification of loss of initial full-length VSG from cells with concomitant release of truncated VSG to the media. Three biological replicates are quantified, and the data are presented as mean ± SD.

Techniques Used: Expressing, Radioactivity, Blocking Assay, Immunoprecipitation, SDS Page

24) Product Images from "Atypical bZIP Domain of Viral Transcription Factor Contributes to Stability of Dimer Formation and Transcriptional Function ▿"

Article Title: Atypical bZIP Domain of Viral Transcription Factor Contributes to Stability of Dimer Formation and Transcriptional Function ▿

Journal: Journal of Virology

doi: 10.1128/JVI.00215-07

Requirement of the proximal CT region for interaction with three ZREs. (A) Proteins were assessed by SOS-PAGE. Abilities of indicated Zta truncation mutants to bind to ZREs were determined by EMSA analysis. In all cases the probe was in excess. Following separation of free and bound ZREs by electrophoresis, locations of the complexes were determined using phosphorimaging, with subsequent quantitation. Duplicate experiments were undertaken, and representative images are shown (B).
Figure Legend Snippet: Requirement of the proximal CT region for interaction with three ZREs. (A) Proteins were assessed by SOS-PAGE. Abilities of indicated Zta truncation mutants to bind to ZREs were determined by EMSA analysis. In all cases the probe was in excess. Following separation of free and bound ZREs by electrophoresis, locations of the complexes were determined using phosphorimaging, with subsequent quantitation. Duplicate experiments were undertaken, and representative images are shown (B).

Techniques Used: Polyacrylamide Gel Electrophoresis, Electrophoresis, Quantitation Assay

Both the proximal and distal parts of the CT region contribute to dimerization. (A) Schematic diagram showing sequence of the truncation mutants spanning the zipper and CT regions. The zipper region is shown as an open box and the CT region as a cross-hatched box. (B) Amounts of translation product generated from wheat germ extract for Zta and Zta truncated mutants used in the dimerization assay were assessed by SDS-PAGE and quantitated by phosphorimaging. (C) Equivalent amounts of each protein were incubated at 37°C and cross-linked by addition of 0.05% glutaraldehyde for 30 min. The reaction was quenched by the addition of glycine, and dimeric proteins were separated on a 10% bis-Tris gel and visualized using phosphorimaging. A representative gel is shown.
Figure Legend Snippet: Both the proximal and distal parts of the CT region contribute to dimerization. (A) Schematic diagram showing sequence of the truncation mutants spanning the zipper and CT regions. The zipper region is shown as an open box and the CT region as a cross-hatched box. (B) Amounts of translation product generated from wheat germ extract for Zta and Zta truncated mutants used in the dimerization assay were assessed by SDS-PAGE and quantitated by phosphorimaging. (C) Equivalent amounts of each protein were incubated at 37°C and cross-linked by addition of 0.05% glutaraldehyde for 30 min. The reaction was quenched by the addition of glycine, and dimeric proteins were separated on a 10% bis-Tris gel and visualized using phosphorimaging. A representative gel is shown.

Techniques Used: Sequencing, Generated, SDS Page, Incubation

25) Product Images from "Functional Interaction between Epstein-Barr Virus Replication Protein Zta and Host DNA Damage Response Protein 53BP1 ▿Functional Interaction between Epstein-Barr Virus Replication Protein Zta and Host DNA Damage Response Protein 53BP1 ▿ †"

Article Title: Functional Interaction between Epstein-Barr Virus Replication Protein Zta and Host DNA Damage Response Protein 53BP1 ▿Functional Interaction between Epstein-Barr Virus Replication Protein Zta and Host DNA Damage Response Protein 53BP1 ▿ †

Journal: Journal of Virology

doi: 10.1128/JVI.00512-09

C-terminal region of Zta is important in 53BP1 binding in vitro. (A) Schematic diagram showing part of the Zta sequence with the location of the Zta termination mutants indicated above the zipper and C-terminal regions. The zipper region is shown by a gray box, and the carboxyl-terminal region is shown by the black box below the sequence. (B) The indicated in vitro-translated proteins were generated in wheat germ extract. Zta and its termination mutants were analyzed by SDS-PAGE on a 12% gel and quantitated after detection using phosphorimaging. (C) Equivalent quantities of in vitro-translated proteins, indicated above each lane, were incubated with GST-53BP1-BRCT-Sepharose or GST, as indicated, and the Zta that associates is shown.
Figure Legend Snippet: C-terminal region of Zta is important in 53BP1 binding in vitro. (A) Schematic diagram showing part of the Zta sequence with the location of the Zta termination mutants indicated above the zipper and C-terminal regions. The zipper region is shown by a gray box, and the carboxyl-terminal region is shown by the black box below the sequence. (B) The indicated in vitro-translated proteins were generated in wheat germ extract. Zta and its termination mutants were analyzed by SDS-PAGE on a 12% gel and quantitated after detection using phosphorimaging. (C) Equivalent quantities of in vitro-translated proteins, indicated above each lane, were incubated with GST-53BP1-BRCT-Sepharose or GST, as indicated, and the Zta that associates is shown.

Techniques Used: Binding Assay, In Vitro, Sequencing, Generated, SDS Page, Incubation

26) Product Images from "The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis *"

Article Title: The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.006940

Loading of FadD32 reaction products onto Pks13 analyzed by SDS-PAGE. Upper , Coomassie Blue staining; lower , phosphorimaging. MW , molecular weight standards. A , loading of acyl-AMP onto Pks13. [ 14 C]C12 acid was used as a precursor. Lanes 1 , absence of
Figure Legend Snippet: Loading of FadD32 reaction products onto Pks13 analyzed by SDS-PAGE. Upper , Coomassie Blue staining; lower , phosphorimaging. MW , molecular weight standards. A , loading of acyl-AMP onto Pks13. [ 14 C]C12 acid was used as a precursor. Lanes 1 , absence of

Techniques Used: SDS Page, Staining, Molecular Weight

Loading of carboxylated chains onto Pks13. SDS-PAGE analyses. Upper , Coomassie Blue staining; lower, phosphorimaging. MW , molecular weight standards. A , loading assays of radiolabeled short chain derivatives to Pks13 wt or Pks13 αβ . Lanes
Figure Legend Snippet: Loading of carboxylated chains onto Pks13. SDS-PAGE analyses. Upper , Coomassie Blue staining; lower, phosphorimaging. MW , molecular weight standards. A , loading assays of radiolabeled short chain derivatives to Pks13 wt or Pks13 αβ . Lanes

Techniques Used: SDS Page, Staining, Molecular Weight

27) Product Images from "Domain swapping between homologous bacterial small RNAs dissects processing and Hfq binding determinants and uncovers an aptamer for conditional RNase E cleavage"

Article Title: Domain swapping between homologous bacterial small RNAs dissects processing and Hfq binding determinants and uncovers an aptamer for conditional RNase E cleavage

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkv1161

Hfq antagonizes cleavage of GlmZ by RapZ/RNase E in vitro . Temporal course of GlmZ processing by RNase E and RapZ in vitro in absence or presence of 150 nM Hfq. Reaction mixtures containing 40 nM α- 32 P-UTP-labelled GlmZ and the proteins designated with plus signs were incubated at 30°C. Samples were removed and reactions were stopped at the indicated times. In the first four lanes GlmZ was incubated for 60 min either alone (lane 1) or together with only one of the three proteins (lanes 2–4) showing that none of these proteins is capable to trigger GlmZ decay when provided alone. Reaction mixtures were separated on denaturing polyacrylamide gels, which were analysed by phosphorimaging. Full-length and processed (additionally labelled with an asterisk) forms of GlmZ are denoted with arrows.
Figure Legend Snippet: Hfq antagonizes cleavage of GlmZ by RapZ/RNase E in vitro . Temporal course of GlmZ processing by RNase E and RapZ in vitro in absence or presence of 150 nM Hfq. Reaction mixtures containing 40 nM α- 32 P-UTP-labelled GlmZ and the proteins designated with plus signs were incubated at 30°C. Samples were removed and reactions were stopped at the indicated times. In the first four lanes GlmZ was incubated for 60 min either alone (lane 1) or together with only one of the three proteins (lanes 2–4) showing that none of these proteins is capable to trigger GlmZ decay when provided alone. Reaction mixtures were separated on denaturing polyacrylamide gels, which were analysed by phosphorimaging. Full-length and processed (additionally labelled with an asterisk) forms of GlmZ are denoted with arrows.

Techniques Used: In Vitro, Incubation

The central stem loop of GlmZ is the decisive element required for RNA cleavage by RNase E and RapZ. In vitro cleavage assays of α- 32 P-UTP-labelled sRNAs using varying concentrations of RNase E-N and a fixed concentration of 150 nM RapZ. sRNAs were generated by in vitro transcription of PCR fragments obtained using the corresponding plasmids (see Supplementary Table S2) as templates. The assays were incubated for 20 min at 30°C, stopped and subsequently separated by denaturing gel electrophoresis and analysed by phosphorimaging. The various sRNA species to be tested were separated on the right half of the gels and their names are given alongside, respectively. Assays using wild-type GlmZ were carried out in parallel and separated alongside (left half of each gel) to allow for direct comparison of cleavage efficiencies. Full-length and processed forms of GlmZ are indicated by arrows.
Figure Legend Snippet: The central stem loop of GlmZ is the decisive element required for RNA cleavage by RNase E and RapZ. In vitro cleavage assays of α- 32 P-UTP-labelled sRNAs using varying concentrations of RNase E-N and a fixed concentration of 150 nM RapZ. sRNAs were generated by in vitro transcription of PCR fragments obtained using the corresponding plasmids (see Supplementary Table S2) as templates. The assays were incubated for 20 min at 30°C, stopped and subsequently separated by denaturing gel electrophoresis and analysed by phosphorimaging. The various sRNA species to be tested were separated on the right half of the gels and their names are given alongside, respectively. Assays using wild-type GlmZ were carried out in parallel and separated alongside (left half of each gel) to allow for direct comparison of cleavage efficiencies. Full-length and processed forms of GlmZ are indicated by arrows.

Techniques Used: In Vitro, Concentration Assay, Generated, Polymerase Chain Reaction, Incubation, Nucleic Acid Electrophoresis

Interaction of GlmY, GlmZ and the various chimeras with Hfq in vitro . EMSAs using α- 32 P-UTP labelled sRNAs and increasing concentrations of purified Hfq protein as indicated. Binding reactions were separated by non-denaturing gel electrophoresis and RNAs were visualized by phosphorimaging. It should be noted that assays using wild-type GlmZ and/or GlmY were carried out in parallel and analysed alongside (data not shown) the respective chimeric sRNA(s) on the same gel to allow for direct comparison of the binding affinities.
Figure Legend Snippet: Interaction of GlmY, GlmZ and the various chimeras with Hfq in vitro . EMSAs using α- 32 P-UTP labelled sRNAs and increasing concentrations of purified Hfq protein as indicated. Binding reactions were separated by non-denaturing gel electrophoresis and RNAs were visualized by phosphorimaging. It should be noted that assays using wild-type GlmZ and/or GlmY were carried out in parallel and analysed alongside (data not shown) the respective chimeric sRNA(s) on the same gel to allow for direct comparison of the binding affinities.

Techniques Used: In Vitro, Purification, Binding Assay, Nucleic Acid Electrophoresis

28) Product Images from "Conserved Intramolecular Disulfide Bond Is Critical to Trafficking and Fate of ATP-binding Cassette (ABC) Transporters ABCB6 and Sulfonylurea Receptor 1 (SUR1)/ABCC8 *"

Article Title: Conserved Intramolecular Disulfide Bond Is Critical to Trafficking and Fate of ATP-binding Cassette (ABC) Transporters ABCB6 and Sulfonylurea Receptor 1 (SUR1)/ABCC8 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.174516

Cys-8 is dispensable for glycosylation but is unstable. A , Cys-8 was substituted with serine or glycine, and the mutant proteins were transiently expressed in NIH3T3 cells. The glycan modification of the mutants was determined by PNGase F sensitivity and analyzed by immunoblotting using anti-V5 antibody. ABCB6 was still glycosylated when Cys-8 was substituted with Ser to make a consensus N -glycosylation motif. B , K562 cells were transduced with plasmids containing IRES-GFP and wild-type ABCB6-, Walker A mutant ( mt )-, C8S-, or C26A-FLAG. Cells were sorted for GFP fluorescence by FACS and analyzed by immunoblotting using anti-FLAG antibody for the expression of ABCB6 proteins. Cysteine mutants C8S and C26A were expressed at low levels. An immunoblot from a single experiment is shown. C , serine was substituted for Cys-8 and/or Cys-26 in ABCB6, or a cysteine residue was inserted between positions 9 and 10 in the C8S mutant ( C8S/C10in ). Endo H sensitivity indicates that all cysteine mutants failed to exit the ER. Cys-10 insertion did not restore the protein expression level nor the impaired ER exit. D , K562 cells stably expressing wild-type ABCB6, C8S, or C26A were labeled with [ 35 S]Met/Cys for 5 min, washed, and chased for the indicated times. Cells were harvested, and immunoprecipitated FLAG-tagged proteins were separated by SDS-PAGE and detected by phosphorimaging. The amount of ABCB6 proteins was analyzed by densitometry and plotted as percentage of ABCB6 protein at 0 h for each construct. Values shown are mean with the S.D. indicated by the error bars from three independent experiments. Unless otherwise noted, all experiments were repeated at least twice. A representative image from one complete experiment is shown.
Figure Legend Snippet: Cys-8 is dispensable for glycosylation but is unstable. A , Cys-8 was substituted with serine or glycine, and the mutant proteins were transiently expressed in NIH3T3 cells. The glycan modification of the mutants was determined by PNGase F sensitivity and analyzed by immunoblotting using anti-V5 antibody. ABCB6 was still glycosylated when Cys-8 was substituted with Ser to make a consensus N -glycosylation motif. B , K562 cells were transduced with plasmids containing IRES-GFP and wild-type ABCB6-, Walker A mutant ( mt )-, C8S-, or C26A-FLAG. Cells were sorted for GFP fluorescence by FACS and analyzed by immunoblotting using anti-FLAG antibody for the expression of ABCB6 proteins. Cysteine mutants C8S and C26A were expressed at low levels. An immunoblot from a single experiment is shown. C , serine was substituted for Cys-8 and/or Cys-26 in ABCB6, or a cysteine residue was inserted between positions 9 and 10 in the C8S mutant ( C8S/C10in ). Endo H sensitivity indicates that all cysteine mutants failed to exit the ER. Cys-10 insertion did not restore the protein expression level nor the impaired ER exit. D , K562 cells stably expressing wild-type ABCB6, C8S, or C26A were labeled with [ 35 S]Met/Cys for 5 min, washed, and chased for the indicated times. Cells were harvested, and immunoprecipitated FLAG-tagged proteins were separated by SDS-PAGE and detected by phosphorimaging. The amount of ABCB6 proteins was analyzed by densitometry and plotted as percentage of ABCB6 protein at 0 h for each construct. Values shown are mean with the S.D. indicated by the error bars from three independent experiments. Unless otherwise noted, all experiments were repeated at least twice. A representative image from one complete experiment is shown.

Techniques Used: Mutagenesis, Modification, Transduction, Fluorescence, FACS, Expressing, Stable Transfection, Labeling, Immunoprecipitation, SDS Page, Construct

29) Product Images from "Conservation of Differential Animal MicroRNA Processing by Drosha and Dicer"

Article Title: Conservation of Differential Animal MicroRNA Processing by Drosha and Dicer

Journal: Frontiers in Molecular Biosciences

doi: 10.3389/fmolb.2021.730006

Purification and activity assays of the fruitfly miRNA processing enzymes. (A) Purification of recombinant Dicer, Drosha/DGCR8 from transfected 293T cells (lanes 1–3) and Loqs-PB from overexpressing bacteria (lanes 4–6). 293T cells were transfectd with plasmids expressing the FLAG-tagged Dicer or FLAG-tagged Drosha and Myc-tagged DGCR8. Following immunoprecipitation with an anti-FLAG antibody, proteins were detected by gel electrophoresis and Coomassie staining. Lane 1: The immunoprecipitate of FLAG-Dicer; lane 2: immunoprecipitate from mock-transfected cells; lane 3: immunoprecipitate of FLAG-Drosha and Myc-DGCR8; lanes 4–6: different elution fractions of His-tagged Loqs-PB after nickle beads purification. Arrows points to the expected fruitfly proteins, and arrowheads the IgG heavy and light chains. Protein markers (in kilodaltons, kD) are indicated on the left. (B) Dicer activity assay. The 32 P-labeled dme-pre-miR-375 was incubated with Dicer alone or together with Loqs-PB at 37°C for 30 min, fractionated on a 12% denaturing gel, and analyzed by phosphorimaging. DNA markers with size of the individual bands in nucleotide (nt) are indicated on the left, and schematics of the substrate and cleavage products shown on the right. (C) Drosha processing assay. The 32 P-labeled dme-pri-let-7 substrate was incubated with Drosha/DCGR8 (Drosha in short) at 37°C for 60 min. Samples were fractionated on a 10% denaturing gel and analyzed by phosphorimaging. Labels are the same as in (B) .
Figure Legend Snippet: Purification and activity assays of the fruitfly miRNA processing enzymes. (A) Purification of recombinant Dicer, Drosha/DGCR8 from transfected 293T cells (lanes 1–3) and Loqs-PB from overexpressing bacteria (lanes 4–6). 293T cells were transfectd with plasmids expressing the FLAG-tagged Dicer or FLAG-tagged Drosha and Myc-tagged DGCR8. Following immunoprecipitation with an anti-FLAG antibody, proteins were detected by gel electrophoresis and Coomassie staining. Lane 1: The immunoprecipitate of FLAG-Dicer; lane 2: immunoprecipitate from mock-transfected cells; lane 3: immunoprecipitate of FLAG-Drosha and Myc-DGCR8; lanes 4–6: different elution fractions of His-tagged Loqs-PB after nickle beads purification. Arrows points to the expected fruitfly proteins, and arrowheads the IgG heavy and light chains. Protein markers (in kilodaltons, kD) are indicated on the left. (B) Dicer activity assay. The 32 P-labeled dme-pre-miR-375 was incubated with Dicer alone or together with Loqs-PB at 37°C for 30 min, fractionated on a 12% denaturing gel, and analyzed by phosphorimaging. DNA markers with size of the individual bands in nucleotide (nt) are indicated on the left, and schematics of the substrate and cleavage products shown on the right. (C) Drosha processing assay. The 32 P-labeled dme-pri-let-7 substrate was incubated with Drosha/DCGR8 (Drosha in short) at 37°C for 60 min. Samples were fractionated on a 10% denaturing gel and analyzed by phosphorimaging. Labels are the same as in (B) .

Techniques Used: Purification, Activity Assay, Recombinant, Transfection, Expressing, Immunoprecipitation, Nucleic Acid Electrophoresis, Staining, Labeling, Incubation

30) Product Images from "A Tyr Residue in the Reverse Transcriptase Domain Can Mimic the Protein-Priming Tyr Residue in the Terminal Protein Domain of a Hepadnavirus P Protein ▿A Tyr Residue in the Reverse Transcriptase Domain Can Mimic the Protein-Priming Tyr Residue in the Terminal Protein Domain of a Hepadnavirus P Protein ▿ †"

Article Title: A Tyr Residue in the Reverse Transcriptase Domain Can Mimic the Protein-Priming Tyr Residue in the Terminal Protein Domain of a Hepadnavirus P Protein ▿A Tyr Residue in the Reverse Transcriptase Domain Can Mimic the Protein-Priming Tyr Residue in the Terminal Protein Domain of a Hepadnavirus P Protein ▿ †

Journal: Journal of Virology

doi: 10.1128/JVI.00482-11

Tyr561 deoxynucleotidylation is not essential for DHBV replication. LMH cells were transfected with plasmids encoding wt DHBV or mutant genomes with Tyr561 in P replaced by Phe (561F) or Ala (561A) and analyzed for replication competence and capsid formation. (A) Replicative DNA intermediates. DNA from cytoplasmic nucleocapsids was analyzed by Southern blotting. (B) Capsid formation. Aliquots from cytoplasmic lysates were subjected to native agarose gel electrophoresis and DHBV capsids were detected by immunoblotting against DHBV core protein. (C) Quantification of replicative intermediates. DNA signals from panel A were quantitated by phosphorimaging, and the sum of signals at the rcDNA and dlDNA positions was normalized to the capsid signal from the same transfection. Numbers represent mean values ± the standard deviation from two independent experiments.
Figure Legend Snippet: Tyr561 deoxynucleotidylation is not essential for DHBV replication. LMH cells were transfected with plasmids encoding wt DHBV or mutant genomes with Tyr561 in P replaced by Phe (561F) or Ala (561A) and analyzed for replication competence and capsid formation. (A) Replicative DNA intermediates. DNA from cytoplasmic nucleocapsids was analyzed by Southern blotting. (B) Capsid formation. Aliquots from cytoplasmic lysates were subjected to native agarose gel electrophoresis and DHBV capsids were detected by immunoblotting against DHBV core protein. (C) Quantification of replicative intermediates. DNA signals from panel A were quantitated by phosphorimaging, and the sum of signals at the rcDNA and dlDNA positions was normalized to the capsid signal from the same transfection. Numbers represent mean values ± the standard deviation from two independent experiments.

Techniques Used: Transfection, Mutagenesis, Southern Blot, Agarose Gel Electrophoresis, Standard Deviation

31) Product Images from "Assaying kinase activity of the TPL-2/NF-κB1 p105/ABIN-2 complex using an optimal peptide substrate"

Article Title: Assaying kinase activity of the TPL-2/NF-κB1 p105/ABIN-2 complex using an optimal peptide substrate

Journal: Biochemical Journal

doi: 10.1042/BCJ20170579

Testing an optimized peptide substrate for TPL-2/NF-κB1 p105/ABIN-2. ( A ) The primary and secondary amino acid preferences for phosphorylation by the recombinant TPL-2/NF-κB1 p105/ABIN-2 complex are shown. ( B ) The sequences and scansite scores for the MKK1 activation loop and optimized TPL-2tide peptide substrates. ( C ) Time-course experiment comparing TPL-2/NF-κB1 p105/ABIN-2 complex phosphorylation of MKK1 and TPL-2tide peptides (50 µM final concentration). Assays were performed with 30 nM the recombinant TPL-2/NF-κB1 p105/ABIN-2 complex in TPL-2 kinase buffer plus 1 mM ATP and 0.02 µCi/µL [γ- 32 P]ATP. Peptides were transferred onto streptavidin-coated membranes, which were then extensively washed. Incorporation of 32 P into peptides was quantified by phosphorimaging. Linear regression was fitted with GraFit version 7.0.3. Values are means ± SD for three replicate reactions. ( D ) Kinase assays as in ( C ) comparing TPL-2tide peptide phosphorylation by wild-type (WT) or kinase-inactive (D270A) TPL-2/NF-κB1 p105/ABIN-2 complex (15 min). ( E and F ) TPL-2 30–404 and TPL-2/NF-κB1 p105/ABIN-2 were titrated (1 : 1.66) in 384-well plates. A substrate solution containing ATP and TPL-2tide ( E ) or S5 peptide ( F ) was then added. The plates were analyzed using a Sciex API6500 ( E ) or API4000 ( F ) Triple Quad with RapidFire™ Technology. The graph shows total peak area counts for the MRM transition 953.6/904.3 Da ( E ; phosphorylated TPL-2tide peptide) and 612.9/120 Da ( F ; phosphorylated S5 peptide) for the indicated enzyme at increasing concentrations. Linear regression was fitted using GraFit software. In E and F , representative graphs from one experiment are shown, including standard deviation of triplicate assays.
Figure Legend Snippet: Testing an optimized peptide substrate for TPL-2/NF-κB1 p105/ABIN-2. ( A ) The primary and secondary amino acid preferences for phosphorylation by the recombinant TPL-2/NF-κB1 p105/ABIN-2 complex are shown. ( B ) The sequences and scansite scores for the MKK1 activation loop and optimized TPL-2tide peptide substrates. ( C ) Time-course experiment comparing TPL-2/NF-κB1 p105/ABIN-2 complex phosphorylation of MKK1 and TPL-2tide peptides (50 µM final concentration). Assays were performed with 30 nM the recombinant TPL-2/NF-κB1 p105/ABIN-2 complex in TPL-2 kinase buffer plus 1 mM ATP and 0.02 µCi/µL [γ- 32 P]ATP. Peptides were transferred onto streptavidin-coated membranes, which were then extensively washed. Incorporation of 32 P into peptides was quantified by phosphorimaging. Linear regression was fitted with GraFit version 7.0.3. Values are means ± SD for three replicate reactions. ( D ) Kinase assays as in ( C ) comparing TPL-2tide peptide phosphorylation by wild-type (WT) or kinase-inactive (D270A) TPL-2/NF-κB1 p105/ABIN-2 complex (15 min). ( E and F ) TPL-2 30–404 and TPL-2/NF-κB1 p105/ABIN-2 were titrated (1 : 1.66) in 384-well plates. A substrate solution containing ATP and TPL-2tide ( E ) or S5 peptide ( F ) was then added. The plates were analyzed using a Sciex API6500 ( E ) or API4000 ( F ) Triple Quad with RapidFire™ Technology. The graph shows total peak area counts for the MRM transition 953.6/904.3 Da ( E ; phosphorylated TPL-2tide peptide) and 612.9/120 Da ( F ; phosphorylated S5 peptide) for the indicated enzyme at increasing concentrations. Linear regression was fitted using GraFit software. In E and F , representative graphs from one experiment are shown, including standard deviation of triplicate assays.

Techniques Used: Recombinant, Activation Assay, Concentration Assay, Software, Standard Deviation

The primary amino acid sequence specificity of TPL-2. The peptide library comprised 198 individual biotinylated peptide mixtures. Each peptide contained a central phosphoracceptor Ser or Thr, flanked by degenerate positions, comprising an equimolar mixture of the 17 amino acids, excluding Cys, Ser and Thr. In each peptide, one position was fixed (fixed residue) with one of the 20 naturally occurring unmodified amino acids, phosphor-Thr (pT) or phosphor-Tyr (pY). Peptides were incubated with 30 nM recombinant TPL-2/NF-κB1 p105/ABIN-2 complex at a final substrate concentration of 50 µM at 30°C for 1 h. Assays were performed in TPL-2 kinase buffer plus 10 µM ATP and 3 nCi/µL [γ- 32 P]ATP in the absence ( A ) or presence ( B ) of C34 TPL-2 inhibitor (10 µM). Following incubation, reactions were transferred onto SAM2 membranes, which were then washed extensively. Incorporation of 32 P into peptides was quantified by phosphorimaging.
Figure Legend Snippet: The primary amino acid sequence specificity of TPL-2. The peptide library comprised 198 individual biotinylated peptide mixtures. Each peptide contained a central phosphoracceptor Ser or Thr, flanked by degenerate positions, comprising an equimolar mixture of the 17 amino acids, excluding Cys, Ser and Thr. In each peptide, one position was fixed (fixed residue) with one of the 20 naturally occurring unmodified amino acids, phosphor-Thr (pT) or phosphor-Tyr (pY). Peptides were incubated with 30 nM recombinant TPL-2/NF-κB1 p105/ABIN-2 complex at a final substrate concentration of 50 µM at 30°C for 1 h. Assays were performed in TPL-2 kinase buffer plus 10 µM ATP and 3 nCi/µL [γ- 32 P]ATP in the absence ( A ) or presence ( B ) of C34 TPL-2 inhibitor (10 µM). Following incubation, reactions were transferred onto SAM2 membranes, which were then washed extensively. Incorporation of 32 P into peptides was quantified by phosphorimaging.

Techniques Used: Sequencing, Incubation, Recombinant, Concentration Assay

32) Product Images from "The bacterial chromatin protein HupA can remodel DNA and associates with the nucleoid in Clostridium difficile"

Article Title: The bacterial chromatin protein HupA can remodel DNA and associates with the nucleoid in Clostridium difficile

Journal: bioRxiv

doi: 10.1101/426809

Dimerization of HupA is independent of DNA binding. (a) Electrophoretic mobility shift assays with increasing concentrations (0.25 −2 µM) of HupA 6xHis and HupA QED 6xHis . Gel shift assays were performed with 2.4 nM radio-labeled ([γ- 32 P] ATP) 29% G+C dsDNA oligonucleotide incubated with HupA for 20 min at room temperature prior to separation. Protein-DNA complexes were analysed on native 8% polyacrylamide gels, vacuum-dried and visualized by phosphorimaging. ssDNA and dsDNA (without protein added, “-”) were used as controls. (b) Quantification of the gel-shift DNA-protein complex by densitometry. Gel shift assays were performed with 2.4 nM radio labelled ([γ- 32 P] ATP) dsDNA oligonucleotides with different 29-71% G+C content and the indicated concentration of HupA 6xHis (red) and HupA QED 6xHis (blue). (c) Elution profiles of HupA 6xHis (red) and HupA QED 6xHis (blue) from size exclusion chromatography. The experiments were performed with purified protein (100 μM) on a Superdex HR 75 10/30 column. The elution position of protein standards of the indicated MW (in kDa) are indicated by vertical grey dashed lines. The elution profiles show a single peak, corresponding to a ∼38 kDa multimer, when compared to the predicted molecular weight of the monomer (11 kDa). No significant difference in the elution profile of the HupA QED 6xHis compared to HupA 6xHis was observed. (d) Western-blot analysis of glutaraldehyde cross-linking of HupA 6xHis and HupA QED 6xHis . 100 ng HupA was incubated with 0%, 0.0006% and 0.006% glutaraldehyde for 30 min at room temperature. The samples were resolved by SDS-PAGE and analysed by immunoblotted with anti-his antibody. Crosslinking between the HupA monomers is observed with the approximate molecular weight of a homo-dimer (∼22 kDa). Additional bands of lower molecular weight HupA are observed (*) that likely represent breakdown products.
Figure Legend Snippet: Dimerization of HupA is independent of DNA binding. (a) Electrophoretic mobility shift assays with increasing concentrations (0.25 −2 µM) of HupA 6xHis and HupA QED 6xHis . Gel shift assays were performed with 2.4 nM radio-labeled ([γ- 32 P] ATP) 29% G+C dsDNA oligonucleotide incubated with HupA for 20 min at room temperature prior to separation. Protein-DNA complexes were analysed on native 8% polyacrylamide gels, vacuum-dried and visualized by phosphorimaging. ssDNA and dsDNA (without protein added, “-”) were used as controls. (b) Quantification of the gel-shift DNA-protein complex by densitometry. Gel shift assays were performed with 2.4 nM radio labelled ([γ- 32 P] ATP) dsDNA oligonucleotides with different 29-71% G+C content and the indicated concentration of HupA 6xHis (red) and HupA QED 6xHis (blue). (c) Elution profiles of HupA 6xHis (red) and HupA QED 6xHis (blue) from size exclusion chromatography. The experiments were performed with purified protein (100 μM) on a Superdex HR 75 10/30 column. The elution position of protein standards of the indicated MW (in kDa) are indicated by vertical grey dashed lines. The elution profiles show a single peak, corresponding to a ∼38 kDa multimer, when compared to the predicted molecular weight of the monomer (11 kDa). No significant difference in the elution profile of the HupA QED 6xHis compared to HupA 6xHis was observed. (d) Western-blot analysis of glutaraldehyde cross-linking of HupA 6xHis and HupA QED 6xHis . 100 ng HupA was incubated with 0%, 0.0006% and 0.006% glutaraldehyde for 30 min at room temperature. The samples were resolved by SDS-PAGE and analysed by immunoblotted with anti-his antibody. Crosslinking between the HupA monomers is observed with the approximate molecular weight of a homo-dimer (∼22 kDa). Additional bands of lower molecular weight HupA are observed (*) that likely represent breakdown products.

Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Incubation, Concentration Assay, Size-exclusion Chromatography, Purification, Molecular Weight, Western Blot, SDS Page

33) Product Images from "Assaying kinase activity of the TPL-2/NF-κB1 p105/ABIN-2 complex using an optimal peptide substrate"

Article Title: Assaying kinase activity of the TPL-2/NF-κB1 p105/ABIN-2 complex using an optimal peptide substrate

Journal: Biochemical Journal

doi: 10.1042/BCJ20170579

Testing an optimized peptide substrate for TPL-2/NF-κB1 p105/ABIN-2. ( A ) The primary and secondary amino acid preferences for phosphorylation by the recombinant TPL-2/NF-κB1 p105/ABIN-2 complex are shown. ( B ) The sequences and scansite scores for the MKK1 activation loop and optimized TPL-2tide peptide substrates. ( C ) Time-course experiment comparing TPL-2/NF-κB1 p105/ABIN-2 complex phosphorylation of MKK1 and TPL-2tide peptides (50 µM final concentration). Assays were performed with 30 nM the recombinant TPL-2/NF-κB1 p105/ABIN-2 complex in TPL-2 kinase buffer plus 1 mM ATP and 0.02 µCi/µL [γ- 32 P]ATP. Peptides were transferred onto streptavidin-coated membranes, which were then extensively washed. Incorporation of 32 P into peptides was quantified by phosphorimaging. Linear regression was fitted with GraFit version 7.0.3. Values are means ± SD for three replicate reactions. ( D ) Kinase assays as in ( C ) comparing TPL-2tide peptide phosphorylation by wild-type (WT) or kinase-inactive (D270A) TPL-2/NF-κB1 p105/ABIN-2 complex (15 min). ( E and F ) TPL-2 30–404 and TPL-2/NF-κB1 p105/ABIN-2 were titrated (1 : 1.66) in 384-well plates. A substrate solution containing ATP and TPL-2tide ( E ) or S5 peptide ( F ) was then added. The plates were analyzed using a Sciex API6500 ( E ) or API4000 ( F ) Triple Quad with RapidFire™ Technology. The graph shows total peak area counts for the MRM transition 953.6/904.3 Da ( E ; phosphorylated TPL-2tide peptide) and 612.9/120 Da ( F ; phosphorylated S5 peptide) for the indicated enzyme at increasing concentrations. Linear regression was fitted using GraFit software. In E and F , representative graphs from one experiment are shown, including standard deviation of triplicate assays.
Figure Legend Snippet: Testing an optimized peptide substrate for TPL-2/NF-κB1 p105/ABIN-2. ( A ) The primary and secondary amino acid preferences for phosphorylation by the recombinant TPL-2/NF-κB1 p105/ABIN-2 complex are shown. ( B ) The sequences and scansite scores for the MKK1 activation loop and optimized TPL-2tide peptide substrates. ( C ) Time-course experiment comparing TPL-2/NF-κB1 p105/ABIN-2 complex phosphorylation of MKK1 and TPL-2tide peptides (50 µM final concentration). Assays were performed with 30 nM the recombinant TPL-2/NF-κB1 p105/ABIN-2 complex in TPL-2 kinase buffer plus 1 mM ATP and 0.02 µCi/µL [γ- 32 P]ATP. Peptides were transferred onto streptavidin-coated membranes, which were then extensively washed. Incorporation of 32 P into peptides was quantified by phosphorimaging. Linear regression was fitted with GraFit version 7.0.3. Values are means ± SD for three replicate reactions. ( D ) Kinase assays as in ( C ) comparing TPL-2tide peptide phosphorylation by wild-type (WT) or kinase-inactive (D270A) TPL-2/NF-κB1 p105/ABIN-2 complex (15 min). ( E and F ) TPL-2 30–404 and TPL-2/NF-κB1 p105/ABIN-2 were titrated (1 : 1.66) in 384-well plates. A substrate solution containing ATP and TPL-2tide ( E ) or S5 peptide ( F ) was then added. The plates were analyzed using a Sciex API6500 ( E ) or API4000 ( F ) Triple Quad with RapidFire™ Technology. The graph shows total peak area counts for the MRM transition 953.6/904.3 Da ( E ; phosphorylated TPL-2tide peptide) and 612.9/120 Da ( F ; phosphorylated S5 peptide) for the indicated enzyme at increasing concentrations. Linear regression was fitted using GraFit software. In E and F , representative graphs from one experiment are shown, including standard deviation of triplicate assays.

Techniques Used: Recombinant, Activation Assay, Concentration Assay, Software, Standard Deviation

The primary amino acid sequence specificity of TPL-2. The peptide library comprised 198 individual biotinylated peptide mixtures. Each peptide contained a central phosphoracceptor Ser or Thr, flanked by degenerate positions, comprising an equimolar mixture of the 17 amino acids, excluding Cys, Ser and Thr. In each peptide, one position was fixed (fixed residue) with one of the 20 naturally occurring unmodified amino acids, phosphor-Thr (pT) or phosphor-Tyr (pY). Peptides were incubated with 30 nM recombinant TPL-2/NF-κB1 p105/ABIN-2 complex at a final substrate concentration of 50 µM at 30°C for 1 h. Assays were performed in TPL-2 kinase buffer plus 10 µM ATP and 3 nCi/µL [γ- 32 P]ATP in the absence ( A ) or presence ( B ) of C34 TPL-2 inhibitor (10 µM). Following incubation, reactions were transferred onto SAM2 membranes, which were then washed extensively. Incorporation of 32 P into peptides was quantified by phosphorimaging.
Figure Legend Snippet: The primary amino acid sequence specificity of TPL-2. The peptide library comprised 198 individual biotinylated peptide mixtures. Each peptide contained a central phosphoracceptor Ser or Thr, flanked by degenerate positions, comprising an equimolar mixture of the 17 amino acids, excluding Cys, Ser and Thr. In each peptide, one position was fixed (fixed residue) with one of the 20 naturally occurring unmodified amino acids, phosphor-Thr (pT) or phosphor-Tyr (pY). Peptides were incubated with 30 nM recombinant TPL-2/NF-κB1 p105/ABIN-2 complex at a final substrate concentration of 50 µM at 30°C for 1 h. Assays were performed in TPL-2 kinase buffer plus 10 µM ATP and 3 nCi/µL [γ- 32 P]ATP in the absence ( A ) or presence ( B ) of C34 TPL-2 inhibitor (10 µM). Following incubation, reactions were transferred onto SAM2 membranes, which were then washed extensively. Incorporation of 32 P into peptides was quantified by phosphorimaging.

Techniques Used: Sequencing, Incubation, Recombinant, Concentration Assay

34) Product Images from "Functional Interaction between Epstein-Barr Virus Replication Protein Zta and Host DNA Damage Response Protein 53BP1 ▿Functional Interaction between Epstein-Barr Virus Replication Protein Zta and Host DNA Damage Response Protein 53BP1 ▿ †"

Article Title: Functional Interaction between Epstein-Barr Virus Replication Protein Zta and Host DNA Damage Response Protein 53BP1 ▿Functional Interaction between Epstein-Barr Virus Replication Protein Zta and Host DNA Damage Response Protein 53BP1 ▿ †

Journal: Journal of Virology

doi: 10.1128/JVI.00512-09

C-terminal region of Zta is important in 53BP1 binding in vitro. (A) Schematic diagram showing part of the Zta sequence with the location of the Zta termination mutants indicated above the zipper and C-terminal regions. The zipper region is shown by a gray box, and the carboxyl-terminal region is shown by the black box below the sequence. (B) The indicated in vitro-translated proteins were generated in wheat germ extract. Zta and its termination mutants were analyzed by SDS-PAGE on a 12% gel and quantitated after detection using phosphorimaging. (C) Equivalent quantities of in vitro-translated proteins, indicated above each lane, were incubated with GST-53BP1-BRCT-Sepharose or GST, as indicated, and the Zta that associates is shown.
Figure Legend Snippet: C-terminal region of Zta is important in 53BP1 binding in vitro. (A) Schematic diagram showing part of the Zta sequence with the location of the Zta termination mutants indicated above the zipper and C-terminal regions. The zipper region is shown by a gray box, and the carboxyl-terminal region is shown by the black box below the sequence. (B) The indicated in vitro-translated proteins were generated in wheat germ extract. Zta and its termination mutants were analyzed by SDS-PAGE on a 12% gel and quantitated after detection using phosphorimaging. (C) Equivalent quantities of in vitro-translated proteins, indicated above each lane, were incubated with GST-53BP1-BRCT-Sepharose or GST, as indicated, and the Zta that associates is shown.

Techniques Used: Binding Assay, In Vitro, Sequencing, Generated, SDS Page, Incubation

35) Product Images from "Unspliced Precursors of NMD-Sensitive ?-Globin Transcripts Exhibit Decreased Steady-State Levels in Erythroid Cells"

Article Title: Unspliced Precursors of NMD-Sensitive ?-Globin Transcripts Exhibit Decreased Steady-State Levels in Erythroid Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038505

Human β-globin pre-mRNAs carrying a nonsense mutation accumulate at low levels in MEL cells. (A) Schematic representation of the test human β-globin constructs stably expressed in MEL cell lines. The closed and open rectangles and lines depict exons, untranslated sequences and introns, respectively. The vertical small arrow represents the position of the nonsense mutation (CAG→UAG) at codon 39 (β39). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m 7 G) and poly(A) tail [(A) n ] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 1-exon 2 sequences (βintron1exon2 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified in each lane, where each number indicates an independent MEL cell line. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin transcripts (see Materials and Methods ). The protected bands corresponding to the human β-globin pre-mRNA and mRNA and mouse α-globin mRNA are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA and pre-mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin (βWT) mRNA from cell line #146 (arbitrary defined as 100%). The values exposed on the graphs (C) and (D) are representative of three independent experiments, and are plotted for each construct showing the mean value and standard deviations. Statistical analysis was performed using the Student's t test (unpaired, two-tailed).
Figure Legend Snippet: Human β-globin pre-mRNAs carrying a nonsense mutation accumulate at low levels in MEL cells. (A) Schematic representation of the test human β-globin constructs stably expressed in MEL cell lines. The closed and open rectangles and lines depict exons, untranslated sequences and introns, respectively. The vertical small arrow represents the position of the nonsense mutation (CAG→UAG) at codon 39 (β39). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m 7 G) and poly(A) tail [(A) n ] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 1-exon 2 sequences (βintron1exon2 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified in each lane, where each number indicates an independent MEL cell line. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin transcripts (see Materials and Methods ). The protected bands corresponding to the human β-globin pre-mRNA and mRNA and mouse α-globin mRNA are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA and pre-mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin (βWT) mRNA from cell line #146 (arbitrary defined as 100%). The values exposed on the graphs (C) and (D) are representative of three independent experiments, and are plotted for each construct showing the mean value and standard deviations. Statistical analysis was performed using the Student's t test (unpaired, two-tailed).

Techniques Used: Mutagenesis, Construct, Stable Transfection, Recombinase Polymerase Amplification, Transfection, Isolation, Two Tailed Test

The decreased β-globin pre-mRNA levels are specific for transcripts carrying NMD-competent nonsense mutations. (A) Schematic representation of the test human β-globin mRNA stably expressed in MEL cell pools. The closed and open rectangles depict exons and untranslated regions, respectively. The vertical small arrows represent the position of the nonsense mutations at codon 26 (GAG→UAG; β26), 39 (CAG→UAG; β39), 62 (GCT→UAG; β62) or 127 (CAG→UAG; β127). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m 7 G) and poly(A) tail [(A) n ] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 2-exon 3 sequences (βintron2exon3 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified above each lane. A 2-fold RNA sample (βWT×2) from MEL cells transfected with the βWT gene was also assayed to demonstrate that the experimental RPA was carried out in probe excess. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin mRNAs (see Materials and Methods ). The protected bands corresponding to the human β-globin and mouse α-globin mRNAs are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin mRNA. (C) The percentage mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (D) Schematic representation of the test human β-globin pre-mRNA stably expressed in MEL cell pools as in ( A ). Localization and length in nucleotides (nt) of the probe comprising part of intron 2 (βintron2 probe) for detection and quantification of the human β-globin pre-mRNA by RPA is shown below the diagram. (E) After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β-globin pre-mRNA and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (see Materials and Methods ). The corresponding protected bands are shown on the right, and their intensities were quantified by phosphorimaging as in ( B ). (F) The percentage pre-mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown, as in ( C ). Statistical analysis was performed using Student's t test (unpaired, two-tailed).
Figure Legend Snippet: The decreased β-globin pre-mRNA levels are specific for transcripts carrying NMD-competent nonsense mutations. (A) Schematic representation of the test human β-globin mRNA stably expressed in MEL cell pools. The closed and open rectangles depict exons and untranslated regions, respectively. The vertical small arrows represent the position of the nonsense mutations at codon 26 (GAG→UAG; β26), 39 (CAG→UAG; β39), 62 (GCT→UAG; β62) or 127 (CAG→UAG; β127). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m 7 G) and poly(A) tail [(A) n ] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 2-exon 3 sequences (βintron2exon3 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified above each lane. A 2-fold RNA sample (βWT×2) from MEL cells transfected with the βWT gene was also assayed to demonstrate that the experimental RPA was carried out in probe excess. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin mRNAs (see Materials and Methods ). The protected bands corresponding to the human β-globin and mouse α-globin mRNAs are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin mRNA. (C) The percentage mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (D) Schematic representation of the test human β-globin pre-mRNA stably expressed in MEL cell pools as in ( A ). Localization and length in nucleotides (nt) of the probe comprising part of intron 2 (βintron2 probe) for detection and quantification of the human β-globin pre-mRNA by RPA is shown below the diagram. (E) After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β-globin pre-mRNA and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (see Materials and Methods ). The corresponding protected bands are shown on the right, and their intensities were quantified by phosphorimaging as in ( B ). (F) The percentage pre-mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown, as in ( C ). Statistical analysis was performed using Student's t test (unpaired, two-tailed).

Techniques Used: Stable Transfection, Recombinase Polymerase Amplification, Transfection, Construct, Isolation, Two Tailed Test

The low levels of the β39 pre-mRNA are not due to the disruption of a regulatory element encompassing codon 39. (A) Schematic representation of the test human β-globin mRNA stably expressed in MEL cell pools. The closed and open rectangles depict exons and untranslated regions, respectively. The vertical small arrow represents the position of the nonsense (CAG→UAG) or missense (CAG→GAG) mutation at codon 39 (β39 and β39missense respectively). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m 7 G) and poly(A) tail [(A) n ] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 2-exon 3 sequences (βintron2exon3 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified above each lane. A 2-fold RNA sample (βWT×2) from MEL cells transfected with the βWT gene was also assayed to demonstrate that the experimental RPA was carried out in probe excess. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin mRNAs (see Materials and Methods ). The protected bands corresponding to the human β-globin and mouse α-globin mRNAs are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin mRNA. (C) The percentage mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (D) Schematic representation of the test human β-globin pre-mRNA stably expressed in MEL cell pools as in ( A ). Localization and length in nucleotides (nt) of the probe comprising part of intron 2 (βintron2 probe) for detection and quantification of the human β-globin pre-mRNA by RPA is shown below the diagram. (E) After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β-globin pre-mRNA and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (see Materials and Methods ). The corresponding protected bands are shown on the right, and their intensities were quantified by phosphorimaging as in ( B ). (F) The percentage pre-mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown, as in (C) .
Figure Legend Snippet: The low levels of the β39 pre-mRNA are not due to the disruption of a regulatory element encompassing codon 39. (A) Schematic representation of the test human β-globin mRNA stably expressed in MEL cell pools. The closed and open rectangles depict exons and untranslated regions, respectively. The vertical small arrow represents the position of the nonsense (CAG→UAG) or missense (CAG→GAG) mutation at codon 39 (β39 and β39missense respectively). Position of initiation (AUG) and termination (UAA) codons, as well as cap structure (m 7 G) and poly(A) tail [(A) n ] are also represented. Localization and length in nucleotides (nt) of the probe comprising intron 2-exon 3 sequences (βintron2exon3 probe) for detection and quantification of the human β-globin RNA by ribonuclease protection assays (RPA) is shown below the diagram. (B) MEL cells were stably transfected with a test human β-globin construct as specified above each lane. A 2-fold RNA sample (βWT×2) from MEL cells transfected with the βWT gene was also assayed to demonstrate that the experimental RPA was carried out in probe excess. After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β- and mouse α-globin mRNAs (see Materials and Methods ). The protected bands corresponding to the human β-globin and mouse α-globin mRNAs are shown on the right, and the corresponding intensities were quantified by phosphorimaging. The level of mRNA from each β-globin allele was normalized to the level of endogenous mouse α-globin in order to control for RNA recovery and erythroid differentiation induction. Normalized values were then calculated as the percentage of wild-type β-globin mRNA. (C) The percentage mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown. Statistical analysis was performed using Student's t test (unpaired, two-tailed). (D) Schematic representation of the test human β-globin pre-mRNA stably expressed in MEL cell pools as in ( A ). Localization and length in nucleotides (nt) of the probe comprising part of intron 2 (βintron2 probe) for detection and quantification of the human β-globin pre-mRNA by RPA is shown below the diagram. (E) After erythroid differentiation induction, steady-state total RNA from either transfected or untransfected (t-) MEL cells was isolated and analysed by RPA using specific probes for human β-globin pre-mRNA and mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (see Materials and Methods ). The corresponding protected bands are shown on the right, and their intensities were quantified by phosphorimaging as in ( B ). (F) The percentage pre-mRNA values were plotted for each construct, and standard deviations from three independent experiments are shown, as in (C) .

Techniques Used: Stable Transfection, Mutagenesis, Recombinase Polymerase Amplification, Transfection, Construct, Isolation, Two Tailed Test

36) Product Images from "The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis *"

Article Title: The Pks13/FadD32 Crosstalk for the Biosynthesis of Mycolic Acids in Mycobacterium tuberculosis *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.006940

Loading of FadD32 reaction products onto Pks13 analyzed by SDS-PAGE. Upper , Coomassie Blue staining; lower , phosphorimaging. MW , molecular weight standards. A , loading of acyl-AMP onto Pks13. [ 14 C]C12 acid was used as a precursor. Lanes 1 , absence of
Figure Legend Snippet: Loading of FadD32 reaction products onto Pks13 analyzed by SDS-PAGE. Upper , Coomassie Blue staining; lower , phosphorimaging. MW , molecular weight standards. A , loading of acyl-AMP onto Pks13. [ 14 C]C12 acid was used as a precursor. Lanes 1 , absence of

Techniques Used: SDS Page, Staining, Molecular Weight

Loading of carboxylated chains onto Pks13. SDS-PAGE analyses. Upper , Coomassie Blue staining; lower, phosphorimaging. MW , molecular weight standards. A , loading assays of radiolabeled short chain derivatives to Pks13 wt or Pks13 αβ . Lanes
Figure Legend Snippet: Loading of carboxylated chains onto Pks13. SDS-PAGE analyses. Upper , Coomassie Blue staining; lower, phosphorimaging. MW , molecular weight standards. A , loading assays of radiolabeled short chain derivatives to Pks13 wt or Pks13 αβ . Lanes

Techniques Used: SDS Page, Staining, Molecular Weight

37) Product Images from "Translational and posttranslational regulation of XIAP by eIF2α and ATF4 promotes ER stress–induced cell death during the unfolded protein response"

Article Title: Translational and posttranslational regulation of XIAP by eIF2α and ATF4 promotes ER stress–induced cell death during the unfolded protein response

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E13-11-0664

PERK signaling attenuates XIAP translation through eIF2α phosphorylation. (A) Wild-type eIF2α S/S MEFs expressing Fv2E-PERK (S/S Fv2E-PERK ) and mutant eIF2α A/A MEFs expressing Fv2E-PERK (A/A Fv2E-PERK ) were treated with 1 nM AP20187, and cell lysates were probed for Fv2E-PERK, phosphorylated eIF2α, or XIAP. Actin served as a loading control. XIAP levels after AP20187 treatment from three different experiments were quantified by densitometry and are shown relative to XIAP levels in untreated cells. (B) Flowchart for metabolic labeling experiments in C and D. (C) Cells expressing Fv2E-PERK were pretreated with AP20187 for the indicated hours and metabolically labeled with 100 μCi of [ 35 S]Met/Cys for 1 h. XIAP was immunoprecipitated, and 35 S-labeled XIAP was visualized by phosphorimaging. Middle, total 35 S-labeled protein levels after AP20187 treatment. (D) eIF2α S/S and eIF2α A/A MEFs were pretreated with 500 nM Tg for 1 h and labeled with 100 μCi of [ 35 S]Met/Cys. XIAP was immunoprecipitated, and 35 S-labeled XIAP was visualized by phosphorimaging. Middle, total 35 S-labeled protein levels after Tg treatment. (C, D) Bottom, Coomassie-stained gel as loading control.
Figure Legend Snippet: PERK signaling attenuates XIAP translation through eIF2α phosphorylation. (A) Wild-type eIF2α S/S MEFs expressing Fv2E-PERK (S/S Fv2E-PERK ) and mutant eIF2α A/A MEFs expressing Fv2E-PERK (A/A Fv2E-PERK ) were treated with 1 nM AP20187, and cell lysates were probed for Fv2E-PERK, phosphorylated eIF2α, or XIAP. Actin served as a loading control. XIAP levels after AP20187 treatment from three different experiments were quantified by densitometry and are shown relative to XIAP levels in untreated cells. (B) Flowchart for metabolic labeling experiments in C and D. (C) Cells expressing Fv2E-PERK were pretreated with AP20187 for the indicated hours and metabolically labeled with 100 μCi of [ 35 S]Met/Cys for 1 h. XIAP was immunoprecipitated, and 35 S-labeled XIAP was visualized by phosphorimaging. Middle, total 35 S-labeled protein levels after AP20187 treatment. (D) eIF2α S/S and eIF2α A/A MEFs were pretreated with 500 nM Tg for 1 h and labeled with 100 μCi of [ 35 S]Met/Cys. XIAP was immunoprecipitated, and 35 S-labeled XIAP was visualized by phosphorimaging. Middle, total 35 S-labeled protein levels after Tg treatment. (C, D) Bottom, Coomassie-stained gel as loading control.

Techniques Used: Expressing, Mutagenesis, Labeling, Metabolic Labelling, Immunoprecipitation, Staining

38) Product Images from "The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu"

Article Title: The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu

Journal: Nature chemical biology

doi: 10.1038/nchembio.1364

Doc phosphorylates EF-Tu at the conserved Thr382 (a). Radiolabeling of EF-Tu by Doc added during (blue) or after (for 15 min; red) ternary complex formation in the presence of γ[ 32 P]-GTP (here and after 3 pmol unless otherwise specified) was analyzed by SDS-PAGE and autoradiography. (b). Radiolabeling of EF-Tu by Doc in the presence of α[ 32 P]-ATP, γ[ 32 P]-ATP, α[ 32 P]-GTP or γ[ 32 P]-GTP. (c). Products of EF-Tu modification (30 s) by Doc in the presence of α[ 32 P]-ATP or γ[ 32 P]-ATP were separated by thin layer chromatography (PEI-cellulose in 0.5M K 2 HPO 4 ) and analyzed by autoradiography. ATP and ADP mobility standards were visualized under UV254 35 , 36 and marked with radioactive spots before phosphorimaging. (d). Phosphorylation of EF-Tu by catalytic mutant of Doc, H66Y. (e). Dipeptide MF synthesis with EF-Tu and EF-Tu T382V in the absence or presence of Doc added before ternary complex formation. (f). Phosphorylation of wild-type EF-Tu and mutant EF-Tu T382V . (g). Doc and γ[ 32 P]-ATP were added to purified EF-Tu, S30 or S100 lysate fractions. A band migrating above the purified EF-Tu corresponds to in vitro aggregated EF-Tu 37 .
Figure Legend Snippet: Doc phosphorylates EF-Tu at the conserved Thr382 (a). Radiolabeling of EF-Tu by Doc added during (blue) or after (for 15 min; red) ternary complex formation in the presence of γ[ 32 P]-GTP (here and after 3 pmol unless otherwise specified) was analyzed by SDS-PAGE and autoradiography. (b). Radiolabeling of EF-Tu by Doc in the presence of α[ 32 P]-ATP, γ[ 32 P]-ATP, α[ 32 P]-GTP or γ[ 32 P]-GTP. (c). Products of EF-Tu modification (30 s) by Doc in the presence of α[ 32 P]-ATP or γ[ 32 P]-ATP were separated by thin layer chromatography (PEI-cellulose in 0.5M K 2 HPO 4 ) and analyzed by autoradiography. ATP and ADP mobility standards were visualized under UV254 35 , 36 and marked with radioactive spots before phosphorimaging. (d). Phosphorylation of EF-Tu by catalytic mutant of Doc, H66Y. (e). Dipeptide MF synthesis with EF-Tu and EF-Tu T382V in the absence or presence of Doc added before ternary complex formation. (f). Phosphorylation of wild-type EF-Tu and mutant EF-Tu T382V . (g). Doc and γ[ 32 P]-ATP were added to purified EF-Tu, S30 or S100 lysate fractions. A band migrating above the purified EF-Tu corresponds to in vitro aggregated EF-Tu 37 .

Techniques Used: Radioactivity, SDS Page, Autoradiography, Modification, Thin Layer Chromatography, Mutagenesis, Purification, In Vitro

Doc inhibits translation by inactivation of ternary complex formation (a). Ribbon representation of the structural superposition of Doc (pdbid 3K33 25 ) onto Fic adenylylase from N. meningitides (NmFic; pdbid 3S6A 1 ). Doc is colored in blue and NmFic in pink, with respective catalytic loops in light and dark green. (b). Superposition of the catalytic loops of Doc (residues H66-R74) and NmFic (in the nucleotide bound conformation; residues H107-R114). The AMPPNP molecule bound to NmFic is shown in black (c) . Luciferase synthesis in a commercially available cell-free translation system was performed in the presence of [ 35 S]-methionine in the absence or presence of Doc and revealed by phosphorimaging. Here and after, full images of gels, TLCs and TLEs are presented in Supplementary Fig. 1 . (d) . Scheme of the assembly of an in vitro translation system using purified components (PK-pyruvate kinase; PEP-phosphoenol pyruvate). Steps at which Doc was added to the reactions in panels (e) and (f) and in Figure 2a , e are depicted in colors. (e) Synthesis of dipeptide MF in the absence or presence of Doc added after (red) or during (blue) ternary complex (TC) formation. (f). Synthesis of tripeptide MFV in the presence or absence of Doc added during initiation (cyan) or ternary (blue) complex formation. Peptides were analyzed by thin layer electrophoresis and autoradiography 14 .
Figure Legend Snippet: Doc inhibits translation by inactivation of ternary complex formation (a). Ribbon representation of the structural superposition of Doc (pdbid 3K33 25 ) onto Fic adenylylase from N. meningitides (NmFic; pdbid 3S6A 1 ). Doc is colored in blue and NmFic in pink, with respective catalytic loops in light and dark green. (b). Superposition of the catalytic loops of Doc (residues H66-R74) and NmFic (in the nucleotide bound conformation; residues H107-R114). The AMPPNP molecule bound to NmFic is shown in black (c) . Luciferase synthesis in a commercially available cell-free translation system was performed in the presence of [ 35 S]-methionine in the absence or presence of Doc and revealed by phosphorimaging. Here and after, full images of gels, TLCs and TLEs are presented in Supplementary Fig. 1 . (d) . Scheme of the assembly of an in vitro translation system using purified components (PK-pyruvate kinase; PEP-phosphoenol pyruvate). Steps at which Doc was added to the reactions in panels (e) and (f) and in Figure 2a , e are depicted in colors. (e) Synthesis of dipeptide MF in the absence or presence of Doc added after (red) or during (blue) ternary complex (TC) formation. (f). Synthesis of tripeptide MFV in the presence or absence of Doc added during initiation (cyan) or ternary (blue) complex formation. Peptides were analyzed by thin layer electrophoresis and autoradiography 14 .

Techniques Used: Luciferase, In Vitro, Purification, Electrophoresis, Autoradiography

39) Product Images from "Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis"

Article Title: Catalytic mechanism of Escherichia coli ribonuclease III: kinetic and inhibitor evidence for the involvement of two magnesium ions in RNA phosphodiester hydrolysis

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki197

N -hydroxyimide inhibition of R1.1 RNA cleavage by RNase III in the presence of Mg 2+ ion. ( A ) Cleavage assay carried out in the presence of increasing concentrations of 2-hydroxy-4H-isoquinoline-1,3-dione (structure shown above gel image). The amount of internally 32 P-labeled R1.1 RNA (synthesized using [α- 32 P]CTP) was 10–40 nmol, and the amount of RNase III was ∼100 fmol. MgCl 2 (10 mM final concentration) was added to initiate the reaction, with a reaction time of 2 min at 37°C. Reactions were electrophoresed in a 15% polyacrylamide gel and visualized by phosphorimaging (see Materials and Methods for additional information). Lane 1 displays a reaction where substrate was incubated with RNase III in the absence of Mg 2+ . Lane 2 displays a reaction where substrate was incubated with Mg 2+ in the absence of RNase III. Lane 3 is the complete reaction, but without added inhibitor. Lanes 4–13 display reactions carried out in the presence of 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 μM inhibitor, respectively. Lane 14 is a control reaction lacking inhibitor, but containing the same amount of ethanol as in the reaction in lane 13. The position of R1.1 RNA is shown on the left and the positions of the two product fragments are on the right. The asterisks indicate positions of small amounts of nonenzymatic breakdown products. ( B ) Cleavage assay carried out in the presence of increasing concentrations of 2-methoxy-1,3(2H,4H)-isoquinolinedione (structure shown above the gel image). The reactions in lanes 1–3 correspond to those of lanes 1–3 in the experiment shown in (A) (see above), and the reactions in lanes 4–8 contain 5, 10, 25, 100 and 250 μM of the compound, respectively. Lane 9 is a control reaction lacking the compound, but containing the same amount of ethanol as in the reaction in lane 8. ( C ) Graphic representation of the inhibitory action of the N -hydroxyimide. The solid triangles represent the percentage inhibition of cleavage by the N -hydroxyimide. The solid squares indicate inhibition by the O -methylated N -hydroxyimide. The open circle represents the effect of ethanol alone on the cleavage reaction.
Figure Legend Snippet: N -hydroxyimide inhibition of R1.1 RNA cleavage by RNase III in the presence of Mg 2+ ion. ( A ) Cleavage assay carried out in the presence of increasing concentrations of 2-hydroxy-4H-isoquinoline-1,3-dione (structure shown above gel image). The amount of internally 32 P-labeled R1.1 RNA (synthesized using [α- 32 P]CTP) was 10–40 nmol, and the amount of RNase III was ∼100 fmol. MgCl 2 (10 mM final concentration) was added to initiate the reaction, with a reaction time of 2 min at 37°C. Reactions were electrophoresed in a 15% polyacrylamide gel and visualized by phosphorimaging (see Materials and Methods for additional information). Lane 1 displays a reaction where substrate was incubated with RNase III in the absence of Mg 2+ . Lane 2 displays a reaction where substrate was incubated with Mg 2+ in the absence of RNase III. Lane 3 is the complete reaction, but without added inhibitor. Lanes 4–13 display reactions carried out in the presence of 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 μM inhibitor, respectively. Lane 14 is a control reaction lacking inhibitor, but containing the same amount of ethanol as in the reaction in lane 13. The position of R1.1 RNA is shown on the left and the positions of the two product fragments are on the right. The asterisks indicate positions of small amounts of nonenzymatic breakdown products. ( B ) Cleavage assay carried out in the presence of increasing concentrations of 2-methoxy-1,3(2H,4H)-isoquinolinedione (structure shown above the gel image). The reactions in lanes 1–3 correspond to those of lanes 1–3 in the experiment shown in (A) (see above), and the reactions in lanes 4–8 contain 5, 10, 25, 100 and 250 μM of the compound, respectively. Lane 9 is a control reaction lacking the compound, but containing the same amount of ethanol as in the reaction in lane 8. ( C ) Graphic representation of the inhibitory action of the N -hydroxyimide. The solid triangles represent the percentage inhibition of cleavage by the N -hydroxyimide. The solid squares indicate inhibition by the O -methylated N -hydroxyimide. The open circle represents the effect of ethanol alone on the cleavage reaction.

Techniques Used: Inhibition, Cleavage Assay, Labeling, Synthesized, Concentration Assay, Incubation, Methylation

E.coli ribonuclease III and the phage T7 substrate, R1.1 RNA. ( A ) Domain structure of the E.coli RNase III polypeptide. The positions of several conserved residues in the nuclease domain are indicated. ( B ) Sequence and structure of R1.1 RNA. The secondary structure is that originally proposed by Dunn and Studier ( 31 ). The cleavage site is indicated by the arrow, with the two products of cleavage 47 and 13 nt in size. ( C ) Gel electrophoretic pattern of R1.1 RNA cleavage, showing the requirement for Mg 2+ . Experimental conditions involved incubation of internally 32 P-labeled R1.1 RNA (synthesized using [α- 32 P]UTP) with RNase III for 5 min at 37°C (see Materials and Methods). The reaction products were separated by electrophoresis in a 15% polyacrylamide gel and then visualized by phosphorimaging. The partial reaction displayed is similar to the gel electrophoretic patterns analyzed in the kinetic analyses (see Results).
Figure Legend Snippet: E.coli ribonuclease III and the phage T7 substrate, R1.1 RNA. ( A ) Domain structure of the E.coli RNase III polypeptide. The positions of several conserved residues in the nuclease domain are indicated. ( B ) Sequence and structure of R1.1 RNA. The secondary structure is that originally proposed by Dunn and Studier ( 31 ). The cleavage site is indicated by the arrow, with the two products of cleavage 47 and 13 nt in size. ( C ) Gel electrophoretic pattern of R1.1 RNA cleavage, showing the requirement for Mg 2+ . Experimental conditions involved incubation of internally 32 P-labeled R1.1 RNA (synthesized using [α- 32 P]UTP) with RNase III for 5 min at 37°C (see Materials and Methods). The reaction products were separated by electrophoresis in a 15% polyacrylamide gel and then visualized by phosphorimaging. The partial reaction displayed is similar to the gel electrophoretic patterns analyzed in the kinetic analyses (see Results).

Techniques Used: Sequencing, Incubation, Labeling, Synthesized, Electrophoresis

2-hydroxy-4H-isoquinoline-1,3-dione inhibition of R1.1 RNA cleavage by RNase III in the presence of Mn 2+ ion. ( A ) Cleavage assay carried out in the presence of increasing concentrations of 2-hydroxy-4H-isoquinoline-1,3-dione (structure shown above gel image). The amount of internally 32 P-labeled R1.1 RNA (synthesized using [α- 32 P]UTP) was 10–40 nmol and the amount of RNase III was 100 fmol. MnCl 2 (2 mM final concentration) was added to initiate the reaction, with a reaction time of 2 min. Reactions were electrophoresed in a 15% polyacrylamide gel and visualized by phosphorimaging (see Materials and Methods for additional information). Lane 1 displays a reaction where substrate was incubated with RNase III in the absence of Mn 2+ . Lane 2 displays a reaction where substrate was incubated with Mn 2+ in the absence of RNase III. Lane 3 is the complete reaction, lacking inhibitor. Lanes 4–15 display reactions carried out in the presence of 0.5, 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 μM inhibitor, respectively. Lane 16 displays a control reaction lacking inhibitor, but containing the same amount of ethanol as in the reaction in lane 15. The position of R1.1 RNA is shown on the left and the positions of the two product fragments are indicated on the right. ( B ) Graphic representation of the inhibitory action of the N -hydroxyimide on the Mn 2+ -supported cleavage reaction. The solid rhombuses (average of three experiments) represent inhibition by the N -hydroxyimide. The open circle represents the effect of ethanol alone on cleavage of substrate (reaction in lane 16).
Figure Legend Snippet: 2-hydroxy-4H-isoquinoline-1,3-dione inhibition of R1.1 RNA cleavage by RNase III in the presence of Mn 2+ ion. ( A ) Cleavage assay carried out in the presence of increasing concentrations of 2-hydroxy-4H-isoquinoline-1,3-dione (structure shown above gel image). The amount of internally 32 P-labeled R1.1 RNA (synthesized using [α- 32 P]UTP) was 10–40 nmol and the amount of RNase III was 100 fmol. MnCl 2 (2 mM final concentration) was added to initiate the reaction, with a reaction time of 2 min. Reactions were electrophoresed in a 15% polyacrylamide gel and visualized by phosphorimaging (see Materials and Methods for additional information). Lane 1 displays a reaction where substrate was incubated with RNase III in the absence of Mn 2+ . Lane 2 displays a reaction where substrate was incubated with Mn 2+ in the absence of RNase III. Lane 3 is the complete reaction, lacking inhibitor. Lanes 4–15 display reactions carried out in the presence of 0.5, 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5 and 25 μM inhibitor, respectively. Lane 16 displays a control reaction lacking inhibitor, but containing the same amount of ethanol as in the reaction in lane 15. The position of R1.1 RNA is shown on the left and the positions of the two product fragments are indicated on the right. ( B ) Graphic representation of the inhibitory action of the N -hydroxyimide on the Mn 2+ -supported cleavage reaction. The solid rhombuses (average of three experiments) represent inhibition by the N -hydroxyimide. The open circle represents the effect of ethanol alone on cleavage of substrate (reaction in lane 16).

Techniques Used: Inhibition, Cleavage Assay, Labeling, Synthesized, Concentration Assay, Incubation

40) Product Images from "The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu"

Article Title: The Fic protein Doc uses an inverted substrate to phosphorylate and inactivate EF-Tu

Journal: Nature chemical biology

doi: 10.1038/nchembio.1364

Doc phosphorylates EF-Tu at the conserved Thr382 (a). Radiolabeling of EF-Tu by Doc added during (blue) or after (for 15 min; red) ternary complex formation in the presence of γ[ 32 P]-GTP (here and after 3 pmol unless otherwise specified) was analyzed by SDS-PAGE and autoradiography. (b). Radiolabeling of EF-Tu by Doc in the presence of α[ 32 P]-ATP, γ[ 32 P]-ATP, α[ 32 P]-GTP or γ[ 32 P]-GTP. (c). Products of EF-Tu modification (30 s) by Doc in the presence of α[ 32 P]-ATP or γ[ 32 P]-ATP were separated by thin layer chromatography (PEI-cellulose in 0.5M K 2 HPO 4 ) and analyzed by autoradiography. ATP and ADP mobility standards were visualized under UV254 35 , 36 and marked with radioactive spots before phosphorimaging. (d). Phosphorylation of EF-Tu by catalytic mutant of Doc, H66Y. (e). Dipeptide MF synthesis with EF-Tu and EF-Tu T382V in the absence or presence of Doc added before ternary complex formation. (f). Phosphorylation of wild-type EF-Tu and mutant EF-Tu T382V . (g). Doc and γ[ 32 P]-ATP were added to purified EF-Tu, S30 or S100 lysate fractions. A band migrating above the purified EF-Tu corresponds to in vitro aggregated EF-Tu 37 .
Figure Legend Snippet: Doc phosphorylates EF-Tu at the conserved Thr382 (a). Radiolabeling of EF-Tu by Doc added during (blue) or after (for 15 min; red) ternary complex formation in the presence of γ[ 32 P]-GTP (here and after 3 pmol unless otherwise specified) was analyzed by SDS-PAGE and autoradiography. (b). Radiolabeling of EF-Tu by Doc in the presence of α[ 32 P]-ATP, γ[ 32 P]-ATP, α[ 32 P]-GTP or γ[ 32 P]-GTP. (c). Products of EF-Tu modification (30 s) by Doc in the presence of α[ 32 P]-ATP or γ[ 32 P]-ATP were separated by thin layer chromatography (PEI-cellulose in 0.5M K 2 HPO 4 ) and analyzed by autoradiography. ATP and ADP mobility standards were visualized under UV254 35 , 36 and marked with radioactive spots before phosphorimaging. (d). Phosphorylation of EF-Tu by catalytic mutant of Doc, H66Y. (e). Dipeptide MF synthesis with EF-Tu and EF-Tu T382V in the absence or presence of Doc added before ternary complex formation. (f). Phosphorylation of wild-type EF-Tu and mutant EF-Tu T382V . (g). Doc and γ[ 32 P]-ATP were added to purified EF-Tu, S30 or S100 lysate fractions. A band migrating above the purified EF-Tu corresponds to in vitro aggregated EF-Tu 37 .

Techniques Used: Radioactivity, SDS Page, Autoradiography, Modification, Thin Layer Chromatography, Mutagenesis, Purification, In Vitro

Doc inhibits translation by inactivation of ternary complex formation (a). Ribbon representation of the structural superposition of Doc (pdbid 3K33 25 ) onto Fic adenylylase from N. meningitides (NmFic; pdbid 3S6A 1 ). Doc is colored in blue and NmFic in pink, with respective catalytic loops in light and dark green. (b). Superposition of the catalytic loops of Doc (residues H66-R74) and NmFic (in the nucleotide bound conformation; residues H107-R114). The AMPPNP molecule bound to NmFic is shown in black (c) . Luciferase synthesis in a commercially available cell-free translation system was performed in the presence of [ 35 S]-methionine in the absence or presence of Doc and revealed by phosphorimaging. Here and after, full images of gels, TLCs and TLEs are presented in Supplementary Fig. 1 . (d) . Scheme of the assembly of an in vitro translation system using purified components (PK-pyruvate kinase; PEP-phosphoenol pyruvate). Steps at which Doc was added to the reactions in panels (e) and (f) and in Figure 2a , e are depicted in colors. (e) Synthesis of dipeptide MF in the absence or presence of Doc added after (red) or during (blue) ternary complex (TC) formation. (f). Synthesis of tripeptide MFV in the presence or absence of Doc added during initiation (cyan) or ternary (blue) complex formation. Peptides were analyzed by thin layer electrophoresis and autoradiography 14 .
Figure Legend Snippet: Doc inhibits translation by inactivation of ternary complex formation (a). Ribbon representation of the structural superposition of Doc (pdbid 3K33 25 ) onto Fic adenylylase from N. meningitides (NmFic; pdbid 3S6A 1 ). Doc is colored in blue and NmFic in pink, with respective catalytic loops in light and dark green. (b). Superposition of the catalytic loops of Doc (residues H66-R74) and NmFic (in the nucleotide bound conformation; residues H107-R114). The AMPPNP molecule bound to NmFic is shown in black (c) . Luciferase synthesis in a commercially available cell-free translation system was performed in the presence of [ 35 S]-methionine in the absence or presence of Doc and revealed by phosphorimaging. Here and after, full images of gels, TLCs and TLEs are presented in Supplementary Fig. 1 . (d) . Scheme of the assembly of an in vitro translation system using purified components (PK-pyruvate kinase; PEP-phosphoenol pyruvate). Steps at which Doc was added to the reactions in panels (e) and (f) and in Figure 2a , e are depicted in colors. (e) Synthesis of dipeptide MF in the absence or presence of Doc added after (red) or during (blue) ternary complex (TC) formation. (f). Synthesis of tripeptide MFV in the presence or absence of Doc added during initiation (cyan) or ternary (blue) complex formation. Peptides were analyzed by thin layer electrophoresis and autoradiography 14 .

Techniques Used: Luciferase, In Vitro, Purification, Electrophoresis, Autoradiography

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    GE Healthcare typhoon fla9500 phosphorimager
    Identifying interacting partners of OXA 1L and assessing mitochondrial protein synthesis in patient fibroblasts Affinity purification mass spectrometry analysis of proteins interacting with OXA1L‐FLAG. Control (HEK293T) or OXA1L‐1 cells expressing OXA1L‐FLAG were solubilised in 1% (w/v) digitonin and incubated with anti‐FLAG affinity gel ( n = 3). Eluates were analysed by label‐free quantitative mass spectrometry (LFQ). Log 2 LFQ intensities were submitted to a modified two‐sided two‐sample t ‐test with significance determined through permutation‐based false discovery rate (FDR) statistics as described in the Materials and Methods. Enriched respiratory complex subunits are colour‐coded, and mtDNA‐encoded subunits are labelled. Full data are provided in Dataset EV1 . Western blot analysis was performed from control (C) and OXA1L patient immortalised fibroblasts (P). The steady‐state level of OXA1L was assessed as were the steady‐state levels of components of the OXPHOS system (NDUFB8, COXII and ATP5β). On a separate Western blot, the levels of the mitoribosomal proteins MRPL3, MRPL45, MRPL11 and MRPS26 were detected. For both the membranes, β‐actin was used as loading control. The dashed line indicates that some lanes were omitted from the figure. Control (C1) and patient (P) fibroblasts were treated with emetine dihydrochloride to inhibit cytosolic translation and mitochondrial protein synthesis analysed by [ 35 S] Met/Cys incorporation with 1 h pulse and either 4 or 8 h chase. Cell lysate (50 μg) was separated through a 15% polyacrylamide gel. The gel was stained with Coomassie blue (CBB) to confirm equal loading. After fixation and drying the signal was visualised using a Typhoon <t>FLA9500</t> <t>PhosphorImager.</t> Signals were ascribed following established migration patterns (Chomyn, 1996 ). Source data are available online for this figure.
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    GE Healthcare phosphorimager
    CPY* is stabilized in sec63-402 . A: CPY* degradation was examined by pulse chase analysis in all new sec63 mutants and the corresponding wildtype. Cells were grown at 30°C to early log phase and labeled with [ 35 S] methionine/cysteine for 2 min, followed by a chase for the indicated times. Cells were lysed and CPY* immunoprecipitated and analysed on 10% gel SDS-gels and detected by autoradiography. B: CPY* was quantified using a <t>phosphorimager;</t> the results of 3 independent experiments are shown in the graph. C: CPY* degradation in sec63-402 at different temperatures; wildtype and sec63-402 were grown at 30°C to early log phase, then cells were transferred to 23°C or 37°C. D: Quantitation of CPY* from the experiments shown in C. Mean values of 2 independent experiments are shown.
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    Identifying interacting partners of OXA 1L and assessing mitochondrial protein synthesis in patient fibroblasts Affinity purification mass spectrometry analysis of proteins interacting with OXA1L‐FLAG. Control (HEK293T) or OXA1L‐1 cells expressing OXA1L‐FLAG were solubilised in 1% (w/v) digitonin and incubated with anti‐FLAG affinity gel ( n = 3). Eluates were analysed by label‐free quantitative mass spectrometry (LFQ). Log 2 LFQ intensities were submitted to a modified two‐sided two‐sample t ‐test with significance determined through permutation‐based false discovery rate (FDR) statistics as described in the Materials and Methods. Enriched respiratory complex subunits are colour‐coded, and mtDNA‐encoded subunits are labelled. Full data are provided in Dataset EV1 . Western blot analysis was performed from control (C) and OXA1L patient immortalised fibroblasts (P). The steady‐state level of OXA1L was assessed as were the steady‐state levels of components of the OXPHOS system (NDUFB8, COXII and ATP5β). On a separate Western blot, the levels of the mitoribosomal proteins MRPL3, MRPL45, MRPL11 and MRPS26 were detected. For both the membranes, β‐actin was used as loading control. The dashed line indicates that some lanes were omitted from the figure. Control (C1) and patient (P) fibroblasts were treated with emetine dihydrochloride to inhibit cytosolic translation and mitochondrial protein synthesis analysed by [ 35 S] Met/Cys incorporation with 1 h pulse and either 4 or 8 h chase. Cell lysate (50 μg) was separated through a 15% polyacrylamide gel. The gel was stained with Coomassie blue (CBB) to confirm equal loading. After fixation and drying the signal was visualised using a Typhoon FLA9500 PhosphorImager. Signals were ascribed following established migration patterns (Chomyn, 1996 ). Source data are available online for this figure.

    Journal: EMBO Molecular Medicine

    Article Title: OXA1L mutations cause mitochondrial encephalopathy and a combined oxidative phosphorylation defect

    doi: 10.15252/emmm.201809060

    Figure Lengend Snippet: Identifying interacting partners of OXA 1L and assessing mitochondrial protein synthesis in patient fibroblasts Affinity purification mass spectrometry analysis of proteins interacting with OXA1L‐FLAG. Control (HEK293T) or OXA1L‐1 cells expressing OXA1L‐FLAG were solubilised in 1% (w/v) digitonin and incubated with anti‐FLAG affinity gel ( n = 3). Eluates were analysed by label‐free quantitative mass spectrometry (LFQ). Log 2 LFQ intensities were submitted to a modified two‐sided two‐sample t ‐test with significance determined through permutation‐based false discovery rate (FDR) statistics as described in the Materials and Methods. Enriched respiratory complex subunits are colour‐coded, and mtDNA‐encoded subunits are labelled. Full data are provided in Dataset EV1 . Western blot analysis was performed from control (C) and OXA1L patient immortalised fibroblasts (P). The steady‐state level of OXA1L was assessed as were the steady‐state levels of components of the OXPHOS system (NDUFB8, COXII and ATP5β). On a separate Western blot, the levels of the mitoribosomal proteins MRPL3, MRPL45, MRPL11 and MRPS26 were detected. For both the membranes, β‐actin was used as loading control. The dashed line indicates that some lanes were omitted from the figure. Control (C1) and patient (P) fibroblasts were treated with emetine dihydrochloride to inhibit cytosolic translation and mitochondrial protein synthesis analysed by [ 35 S] Met/Cys incorporation with 1 h pulse and either 4 or 8 h chase. Cell lysate (50 μg) was separated through a 15% polyacrylamide gel. The gel was stained with Coomassie blue (CBB) to confirm equal loading. After fixation and drying the signal was visualised using a Typhoon FLA9500 PhosphorImager. Signals were ascribed following established migration patterns (Chomyn, 1996 ). Source data are available online for this figure.

    Article Snippet: Signals were detected using the Typhoon FLA9500 Phosphorimager and ImageQuant software (GE Healthcare).

    Techniques: Affinity Purification, Mass Spectrometry, Expressing, Incubation, Modification, Western Blot, Staining, Migration

    CPY* is stabilized in sec63-402 . A: CPY* degradation was examined by pulse chase analysis in all new sec63 mutants and the corresponding wildtype. Cells were grown at 30°C to early log phase and labeled with [ 35 S] methionine/cysteine for 2 min, followed by a chase for the indicated times. Cells were lysed and CPY* immunoprecipitated and analysed on 10% gel SDS-gels and detected by autoradiography. B: CPY* was quantified using a phosphorimager; the results of 3 independent experiments are shown in the graph. C: CPY* degradation in sec63-402 at different temperatures; wildtype and sec63-402 were grown at 30°C to early log phase, then cells were transferred to 23°C or 37°C. D: Quantitation of CPY* from the experiments shown in C. Mean values of 2 independent experiments are shown.

    Journal: PLoS ONE

    Article Title: The Sec63p J-Domain Is Required for ERAD of Soluble Proteins in Yeast

    doi: 10.1371/journal.pone.0082058

    Figure Lengend Snippet: CPY* is stabilized in sec63-402 . A: CPY* degradation was examined by pulse chase analysis in all new sec63 mutants and the corresponding wildtype. Cells were grown at 30°C to early log phase and labeled with [ 35 S] methionine/cysteine for 2 min, followed by a chase for the indicated times. Cells were lysed and CPY* immunoprecipitated and analysed on 10% gel SDS-gels and detected by autoradiography. B: CPY* was quantified using a phosphorimager; the results of 3 independent experiments are shown in the graph. C: CPY* degradation in sec63-402 at different temperatures; wildtype and sec63-402 were grown at 30°C to early log phase, then cells were transferred to 23°C or 37°C. D: Quantitation of CPY* from the experiments shown in C. Mean values of 2 independent experiments are shown.

    Article Snippet: The signal was detected by autoradiography on a phosphorimager (Typhoon, GE Healthcare) and quantitation was performed with ImageQuant TL (GE Healthcare).

    Techniques: Pulse Chase, Labeling, Immunoprecipitation, Autoradiography, Quantitation Assay

    Substitutions in CRV of L protein result in defects in mRNA cap formation. (A) Schematic of the 3′ end of the VSV genome and the products of in vitro RNA synthesis. Leader RNA (Le+) and N mRNAs were detected by primer extension using primers denoted x and y , respectively. (B) Products were analyzed on denaturing 6% polyacrylamide gels and detected using a PhosphorImager. The size of the RT product is shown. Where indicated, samples were treated with TAP to hydrolyze the cap structure. Under these conditions, RT cannot extend onto the cap, thus yielding a 1-nt-shorter product. rL, recombinant L. (C) Viral mRNA was synthesized as for panel A, except 15 μCi of [α- 32 P]GTP was incorporated into the reaction. Purified RNAs were digested with TAP and analyzed by TLC. Plates were dried, and spots were visualized using a PhosphorImager. Migration of markers 7 m Gp and Gp and the chromatographic origin are shown. (D) Quantitative analysis performed on three independent experiments. Released Gp (mean ± standard deviation) was expressed as a percentage of the total released Gp from wt L.

    Journal: Journal of Virology

    Article Title: A Conserved Motif in Region V of the Large Polymerase Proteins of Nonsegmented Negative-Sense RNA Viruses That Is Essential for mRNA Capping ▿

    doi: 10.1128/JVI.02107-07

    Figure Lengend Snippet: Substitutions in CRV of L protein result in defects in mRNA cap formation. (A) Schematic of the 3′ end of the VSV genome and the products of in vitro RNA synthesis. Leader RNA (Le+) and N mRNAs were detected by primer extension using primers denoted x and y , respectively. (B) Products were analyzed on denaturing 6% polyacrylamide gels and detected using a PhosphorImager. The size of the RT product is shown. Where indicated, samples were treated with TAP to hydrolyze the cap structure. Under these conditions, RT cannot extend onto the cap, thus yielding a 1-nt-shorter product. rL, recombinant L. (C) Viral mRNA was synthesized as for panel A, except 15 μCi of [α- 32 P]GTP was incorporated into the reaction. Purified RNAs were digested with TAP and analyzed by TLC. Plates were dried, and spots were visualized using a PhosphorImager. Migration of markers 7 m Gp and Gp and the chromatographic origin are shown. (D) Quantitative analysis performed on three independent experiments. Released Gp (mean ± standard deviation) was expressed as a percentage of the total released Gp from wt L.

    Article Snippet: Quantitative analysis was performed by using a PhosphorImager (GE Healthcare, Typhoon) and ImageQuant TL software (GE Healthcare, Piscataway, NJ).

    Techniques: In Vitro, Recombinant, Synthesized, Purification, Thin Layer Chromatography, Migration, Standard Deviation

    Reconstitution of VSV mRNA synthesis in vitro. (A) Virus (V), ribonucleoprotein cores (R), N RNA template (T), and BV-expressed recombinant L (rL) and P (rP) proteins were purified and analyzed by SDS-PAGE. An image of the Coomassie blue-stained gel is shown. Lane 1, molecular weight standards (Mr); lane 2, 5 μg of purified VSV; lane 3, 3 μg of RNP; lane 4, 1.5 μg of N RNA template; lane 5, 1 μg of rL; lane 6, 0.5 μg of rP. (B) In vitro transcription reactions using 5, 10, and 15 μg of purified RNP (lanes 1 to 3) or reconstituted from 1 μg of N RNA, 0.5 μg of P, and 0.5, 1, or 2 μg L (lanes 4 to 9) were performed in the presence of [α- 32 P]GTP. Purified RNA was analyzed on acid-agarose gels and detected using a PhosphorImager. (C) TLC analysis of the cap. Transcription reactions were performed in the presence of SAM or SAH and [α- 32 P]GTP. Purified RNAs were digested with TAP, and the products were analyzed by TLC. Plates were dried, and spots were visualized with a PhosphorImager. The migration of markers 7 m Gp and Gp is shown.

    Journal: Journal of Virology

    Article Title: A Conserved Motif in Region V of the Large Polymerase Proteins of Nonsegmented Negative-Sense RNA Viruses That Is Essential for mRNA Capping ▿

    doi: 10.1128/JVI.02107-07

    Figure Lengend Snippet: Reconstitution of VSV mRNA synthesis in vitro. (A) Virus (V), ribonucleoprotein cores (R), N RNA template (T), and BV-expressed recombinant L (rL) and P (rP) proteins were purified and analyzed by SDS-PAGE. An image of the Coomassie blue-stained gel is shown. Lane 1, molecular weight standards (Mr); lane 2, 5 μg of purified VSV; lane 3, 3 μg of RNP; lane 4, 1.5 μg of N RNA template; lane 5, 1 μg of rL; lane 6, 0.5 μg of rP. (B) In vitro transcription reactions using 5, 10, and 15 μg of purified RNP (lanes 1 to 3) or reconstituted from 1 μg of N RNA, 0.5 μg of P, and 0.5, 1, or 2 μg L (lanes 4 to 9) were performed in the presence of [α- 32 P]GTP. Purified RNA was analyzed on acid-agarose gels and detected using a PhosphorImager. (C) TLC analysis of the cap. Transcription reactions were performed in the presence of SAM or SAH and [α- 32 P]GTP. Purified RNAs were digested with TAP, and the products were analyzed by TLC. Plates were dried, and spots were visualized with a PhosphorImager. The migration of markers 7 m Gp and Gp is shown.

    Article Snippet: Quantitative analysis was performed by using a PhosphorImager (GE Healthcare, Typhoon) and ImageQuant TL software (GE Healthcare, Piscataway, NJ).

    Techniques: In Vitro, Recombinant, Purification, SDS Page, Staining, Molecular Weight, Thin Layer Chromatography, Migration

    DIDS disrupts the DNA binding activity of eh Rad51. (A) Time course analysis of eh Rad51 ATPase activity in the presence or absence of DIDS (67 μM), with and without ϕX174 ssDNA or linearized ϕX174 dsDNA. Reactions were stopped with the addition of EDTA at the indicated times prior to separation with thin-layer chromatography and phosphorimager analysis. (B) eh Rad51 (7 μM) incubated with 32 P-radiolabeled ssDNA and increasing amounts of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM; lanes 3–8 respectively). Lane 1 contained no protein or DIDS. Lane 2 contained no DIDS. Lane 9 contained 40 μM DIDS without eh Rad51. (C) eh Rad51 (35 μM) incubated with the 32 P-radiolabled dsDNA and increasing concentrations of DIDS (20 μM, 40 μM, 80 μM, 100 μM, 150 μM, 200 μM; lanes 3–8 respectively). Lane 1 lacked protein and DIDS, lane 2 contained no DIDS and lane 9 contained 200 μM DIDS and no protein. (D) eh Rad51 (7 μM) was incubated with radiolabeled ssDNA in the absence (lane 3) and presence of increasing concentrations of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 40 μM; lanes 4–9) prior to the addition of DNase I. The reaction were deproteinized, and the products were separated using non-denaturing PAGE. Lane 1 contained radiolabeled ssDNA alone, lane 2 contained DNase with radiolabeled ssDNA, and lane 10 contained radiolabeled ssDNA in the presence of 40 μM DIDS and DNase I. Error bars represent SEM (n = 3).

    Journal: Molecular and biochemical parasitology

    Article Title: Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase

    doi: 10.1016/j.molbiopara.2016.09.001

    Figure Lengend Snippet: DIDS disrupts the DNA binding activity of eh Rad51. (A) Time course analysis of eh Rad51 ATPase activity in the presence or absence of DIDS (67 μM), with and without ϕX174 ssDNA or linearized ϕX174 dsDNA. Reactions were stopped with the addition of EDTA at the indicated times prior to separation with thin-layer chromatography and phosphorimager analysis. (B) eh Rad51 (7 μM) incubated with 32 P-radiolabeled ssDNA and increasing amounts of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM; lanes 3–8 respectively). Lane 1 contained no protein or DIDS. Lane 2 contained no DIDS. Lane 9 contained 40 μM DIDS without eh Rad51. (C) eh Rad51 (35 μM) incubated with the 32 P-radiolabled dsDNA and increasing concentrations of DIDS (20 μM, 40 μM, 80 μM, 100 μM, 150 μM, 200 μM; lanes 3–8 respectively). Lane 1 lacked protein and DIDS, lane 2 contained no DIDS and lane 9 contained 200 μM DIDS and no protein. (D) eh Rad51 (7 μM) was incubated with radiolabeled ssDNA in the absence (lane 3) and presence of increasing concentrations of DIDS (5 μM, 10 μM, 15 μM, 20 μM, 30 μM, and 40 μM; lanes 4–9) prior to the addition of DNase I. The reaction were deproteinized, and the products were separated using non-denaturing PAGE. Lane 1 contained radiolabeled ssDNA alone, lane 2 contained DNase with radiolabeled ssDNA, and lane 10 contained radiolabeled ssDNA in the presence of 40 μM DIDS and DNase I. Error bars represent SEM (n = 3).

    Article Snippet: The gels were dried on Whatman cellulose chromatography paper (Sigma-Aldrich), and analyzed using a phosphorimager (GE Healthcare Life Sciences Typhoon FLA 7000).

    Techniques: Binding Assay, Activity Assay, Thin Layer Chromatography, Incubation, Polyacrylamide Gel Electrophoresis

    mHop2-Mnd1 physically interacts with eh Rad51 and enhances eh Rad51 homologous DNA pairing activity. (A) eh Rad51 was incubated with mHop2-Mnd1 (H2M1) immobilized on Affi-gel or bovine serum albumin (BSA) immobilized on Affi-gel. The supernatant was removed, the beads were washed, and interacting proteins eluted with SDS. Aliquots of the supernatant (S), wash (W), and eluate (E) for Affi-mHop2-Mnd1 (lanes 2–4) and Affi-BSA (lanes 5–7) were separated using 12% SDS-PAGE and stained with Coomassie blue. Lane 1 contained molecular weight markers (sizes indicated). (B) Time course analysis of eh Rad51 incubated with 32 P-radiolabeled ssDNA for filament formation in the absence (lanes 2–6) or presence (lanes 8–12) of calcium, followed by the addition of mHop2-Mnd1 and supercoiled duplex DNA. Reactions were deproteinized at the indicated times, subjected to agarose gel electrophoresis, and analyzed using a phosphorimager. Lane 1 contained mHop2-Mnd1 and no eh Rad51, and lane 7 contained mHop2-Mnd1 and calcium but no eh Rad51. Results are represented as percent D-loop formed from three independent experiments. Error bars represent SEM.

    Journal: Molecular and biochemical parasitology

    Article Title: Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase

    doi: 10.1016/j.molbiopara.2016.09.001

    Figure Lengend Snippet: mHop2-Mnd1 physically interacts with eh Rad51 and enhances eh Rad51 homologous DNA pairing activity. (A) eh Rad51 was incubated with mHop2-Mnd1 (H2M1) immobilized on Affi-gel or bovine serum albumin (BSA) immobilized on Affi-gel. The supernatant was removed, the beads were washed, and interacting proteins eluted with SDS. Aliquots of the supernatant (S), wash (W), and eluate (E) for Affi-mHop2-Mnd1 (lanes 2–4) and Affi-BSA (lanes 5–7) were separated using 12% SDS-PAGE and stained with Coomassie blue. Lane 1 contained molecular weight markers (sizes indicated). (B) Time course analysis of eh Rad51 incubated with 32 P-radiolabeled ssDNA for filament formation in the absence (lanes 2–6) or presence (lanes 8–12) of calcium, followed by the addition of mHop2-Mnd1 and supercoiled duplex DNA. Reactions were deproteinized at the indicated times, subjected to agarose gel electrophoresis, and analyzed using a phosphorimager. Lane 1 contained mHop2-Mnd1 and no eh Rad51, and lane 7 contained mHop2-Mnd1 and calcium but no eh Rad51. Results are represented as percent D-loop formed from three independent experiments. Error bars represent SEM.

    Article Snippet: The gels were dried on Whatman cellulose chromatography paper (Sigma-Aldrich), and analyzed using a phosphorimager (GE Healthcare Life Sciences Typhoon FLA 7000).

    Techniques: Activity Assay, Incubation, SDS Page, Staining, Molecular Weight, Agarose Gel Electrophoresis

    eh Rad51 hydrolyzes ATP and binds DNA. (A) Purified recombinant eh Rad51 (0.5 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. (B) Time course analysis of eh Rad51 ATPase activity in the absence and presence of ϕX174 ssDNA or linearized ϕX174 dsDNA. (C) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled ssDNA, and were resolved on a 12% polyacrylamide gel. (D) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled dsDNA. The samples were resolved on a 12% polyacrylamide gel. The results for B, C and D were quantified using a phosphorimager and graphed. Lane 1 for C and D contained no protein, and lane 7 for C and D was treated with SDS/PK (S/P). Error bars represent SEM (n = 3).

    Journal: Molecular and biochemical parasitology

    Article Title: Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase

    doi: 10.1016/j.molbiopara.2016.09.001

    Figure Lengend Snippet: eh Rad51 hydrolyzes ATP and binds DNA. (A) Purified recombinant eh Rad51 (0.5 μg) on a 12% SDS-polyacrylamide gel stained with Coomassie blue. (B) Time course analysis of eh Rad51 ATPase activity in the absence and presence of ϕX174 ssDNA or linearized ϕX174 dsDNA. (C) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled ssDNA, and were resolved on a 12% polyacrylamide gel. (D) Increasing concentrations of eh Rad51 (lanes 2–6) were incubated with 32 P-labeled dsDNA. The samples were resolved on a 12% polyacrylamide gel. The results for B, C and D were quantified using a phosphorimager and graphed. Lane 1 for C and D contained no protein, and lane 7 for C and D was treated with SDS/PK (S/P). Error bars represent SEM (n = 3).

    Article Snippet: The gels were dried on Whatman cellulose chromatography paper (Sigma-Aldrich), and analyzed using a phosphorimager (GE Healthcare Life Sciences Typhoon FLA 7000).

    Techniques: Purification, Recombinant, Staining, Activity Assay, Incubation, Labeling

    eh Rad51 forms a nucleoprotein filament. (A) Time course analysis for eh Rad51 presynaptic filament formation on 32 P-labeled ssDNA (ss) in the presence of DNase I (lanes 3–7). (B) Presynaptic filament formation in the absence and presence of ATP, AMP-PNP, ATP-γ-S, or ADP, as indicated. Samples were resolved on native polyacrylamide gels and analyzed using a phosphorimager. For both (A) and (B), lane 1 contained only 32 P-labeled ssDNA, and lane 2 contained 32 P-labeled ssDNA and DNase I. The results are from three independent experiments and are presented as percent protection. Error bars represent SEM. Deg, degraded 32 P-labeled ssDNA.

    Journal: Molecular and biochemical parasitology

    Article Title: Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase

    doi: 10.1016/j.molbiopara.2016.09.001

    Figure Lengend Snippet: eh Rad51 forms a nucleoprotein filament. (A) Time course analysis for eh Rad51 presynaptic filament formation on 32 P-labeled ssDNA (ss) in the presence of DNase I (lanes 3–7). (B) Presynaptic filament formation in the absence and presence of ATP, AMP-PNP, ATP-γ-S, or ADP, as indicated. Samples were resolved on native polyacrylamide gels and analyzed using a phosphorimager. For both (A) and (B), lane 1 contained only 32 P-labeled ssDNA, and lane 2 contained 32 P-labeled ssDNA and DNase I. The results are from three independent experiments and are presented as percent protection. Error bars represent SEM. Deg, degraded 32 P-labeled ssDNA.

    Article Snippet: The gels were dried on Whatman cellulose chromatography paper (Sigma-Aldrich), and analyzed using a phosphorimager (GE Healthcare Life Sciences Typhoon FLA 7000).

    Techniques: Labeling

    eh Rad51 mediates oligonucleotide DNA strand exchange. (A) Schematic of oligonucleotide DNA strand exchange assay; * represents 32 P-radiolabeled oligonu-cleotide. (B) eh Rad51 (0.25 μM, lane 2; 0.5 μM, lane 3; 0.75 μM, lane 4; 1 μM, lanes 5–9) was incubated with unlabeled ssDNA in the presence (lanes 2–5) or absence (lane 6) of ATP, or in the presence of ATP-γ-S (lane 7), AMP-PNP (lane 8), or ADP (lane 9) followed by the addition of radiolabeled dsDNA complex. Lane 1 contained no protein. Reaction products were deproteinized and separated on a 12% non-denaturing polyacrylamide gel followed by analysis of the gels using a phosphorimager. Results from three separate experiments were graphed with DNA strand exchange activity presented as percent product of the displaced 32 P-ssDNA. Error bars represent SEM.

    Journal: Molecular and biochemical parasitology

    Article Title: Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase

    doi: 10.1016/j.molbiopara.2016.09.001

    Figure Lengend Snippet: eh Rad51 mediates oligonucleotide DNA strand exchange. (A) Schematic of oligonucleotide DNA strand exchange assay; * represents 32 P-radiolabeled oligonu-cleotide. (B) eh Rad51 (0.25 μM, lane 2; 0.5 μM, lane 3; 0.75 μM, lane 4; 1 μM, lanes 5–9) was incubated with unlabeled ssDNA in the presence (lanes 2–5) or absence (lane 6) of ATP, or in the presence of ATP-γ-S (lane 7), AMP-PNP (lane 8), or ADP (lane 9) followed by the addition of radiolabeled dsDNA complex. Lane 1 contained no protein. Reaction products were deproteinized and separated on a 12% non-denaturing polyacrylamide gel followed by analysis of the gels using a phosphorimager. Results from three separate experiments were graphed with DNA strand exchange activity presented as percent product of the displaced 32 P-ssDNA. Error bars represent SEM.

    Article Snippet: The gels were dried on Whatman cellulose chromatography paper (Sigma-Aldrich), and analyzed using a phosphorimager (GE Healthcare Life Sciences Typhoon FLA 7000).

    Techniques: Incubation, Activity Assay

    eh Rad51 oligonucleotide DNA strand exchange activity is stimulated by calcium and mHop2-Mnd1. (A) Time course analysis of eh Rad51 in the absence (top row) or presence of calcium (Ca 2+ , second row), mHop2-Mnd1 (H2M1; third row), or both calcium and mHop2-Mnd1 (Ca 2+ + H2M1, bottom row). Lane 1 for each condition contained no eh Rad51. Reactions were deproteinized at the indicated time points, resolved using native polyacrylamide gel electrophoresis, and analyzed using a phosphorimager. (B) Results from three independent experiments of each condition were quantified and graphed as percent product. Error bars represent SEM.

    Journal: Molecular and biochemical parasitology

    Article Title: Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase

    doi: 10.1016/j.molbiopara.2016.09.001

    Figure Lengend Snippet: eh Rad51 oligonucleotide DNA strand exchange activity is stimulated by calcium and mHop2-Mnd1. (A) Time course analysis of eh Rad51 in the absence (top row) or presence of calcium (Ca 2+ , second row), mHop2-Mnd1 (H2M1; third row), or both calcium and mHop2-Mnd1 (Ca 2+ + H2M1, bottom row). Lane 1 for each condition contained no eh Rad51. Reactions were deproteinized at the indicated time points, resolved using native polyacrylamide gel electrophoresis, and analyzed using a phosphorimager. (B) Results from three independent experiments of each condition were quantified and graphed as percent product. Error bars represent SEM.

    Article Snippet: The gels were dried on Whatman cellulose chromatography paper (Sigma-Aldrich), and analyzed using a phosphorimager (GE Healthcare Life Sciences Typhoon FLA 7000).

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis

    Calcium stimulates the homologous DNA pairing activity of eh Rad51. (A) Schematic of D-loop formation assay. * represents 32 P-radiolabeled oligonucleotide. (B) eh Rad51 was incubated with 32 P-ssDNA (ss) in the absence (lanes 2–6) or presence of calcium (lanes 8–12). Reactions were initiated by the addition of supercoiled duplex DNA (sc) and were deproteinized at the indicated times. Lane 1 contained no protein, and lane 7 contained calcium and no protein. Reaction products from three independent experiments were analyzed by resolving with agarose gel electrophoresis and quantified using a phosphorimager. Error bars represent SEM.

    Journal: Molecular and biochemical parasitology

    Article Title: Characterization of the recombination activities of the Entamoeba histolytica Rad51 recombinase

    doi: 10.1016/j.molbiopara.2016.09.001

    Figure Lengend Snippet: Calcium stimulates the homologous DNA pairing activity of eh Rad51. (A) Schematic of D-loop formation assay. * represents 32 P-radiolabeled oligonucleotide. (B) eh Rad51 was incubated with 32 P-ssDNA (ss) in the absence (lanes 2–6) or presence of calcium (lanes 8–12). Reactions were initiated by the addition of supercoiled duplex DNA (sc) and were deproteinized at the indicated times. Lane 1 contained no protein, and lane 7 contained calcium and no protein. Reaction products from three independent experiments were analyzed by resolving with agarose gel electrophoresis and quantified using a phosphorimager. Error bars represent SEM.

    Article Snippet: The gels were dried on Whatman cellulose chromatography paper (Sigma-Aldrich), and analyzed using a phosphorimager (GE Healthcare Life Sciences Typhoon FLA 7000).

    Techniques: Activity Assay, Tube Formation Assay, Incubation, Agarose Gel Electrophoresis