Structured Review

GE Healthcare α 32 p gdp
Etf-2 has a GAP TBC-like dual catalytic finger motif that directly binds to RAB5-GTP but lacks RAB5 GAP activity. ( A , Upper ) Putative domain structures of Etf-2. Numbers indicate amino acid positions. Q, glutamine finger; R, arginine finger; the blue boxed domain is the putative T4SS signal. ( Lower ) Amino acid sequence alignment of Etf-2 with TBC domains of RABGAP5 and Shigella VirA that have RAB1 GAP activity. Etf-2 has the GAP TBC-like dual (Arg and Glu) catalytic finger motif (red) near its C terminus. ( B ) Direct interaction of full-length Etf-2 with RAB5-WT, -DN, and -CA was tested using the yeast two-hybrid assay. Twenty independent colonies were tested on SD/−Leu/−Trp double-dropout (DDO) and SD/−Leu/−Trp/−His/−Ade quadruple-dropout (QDO) agar plates. Growth on QDO plates indicated a specific interaction between Etf-2 and RAB5-WT or -CA. ( C ) MST assay with purified proteins demonstrating binding of RAB5A to Etf-2 ∆HY in a GTP-dependent manner. RAB5A was fluorescently labeled with Tris-nitrilotriacetic acid at fixed concentrations ranging from 5 to 200 nM and was preincubated with GTPγS or GDPβS; binding was assessed with unlabeled Etf-2 ∆HY from the low picomolar range to 100 µM. RAB5A binding to Etf-2 ∆HY was observed only in RAB5A-GTPγS samples. The determined K d value is shown. Data are shown as mean ± SD ( n = 4). ( D ) GAP activity of Etf-2 ∆HY . GST-RAB5 that had been affinity-purified on GST resin was preloaded with [α- 32 P]GTP followed by a GTP hydrolysis reaction in the absence or presence of RABGAP5 or Etf-2 ∆HY . Samples were taken at the indicated times and subjected to TLC followed by autoradiography and PhosphorImager analysis to visualize and quantify the radiolabeled hydrolysis product ( 32 <t>P-GDP)</t> from substrate ( 32 P-GTP) as indicated. The percentage values indicate the amount of GDP produced from GTP.
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1) Product Images from "Ehrlichia type IV secretion system effector Etf-2 binds to active RAB5 and delays endosome maturation"

Article Title: Ehrlichia type IV secretion system effector Etf-2 binds to active RAB5 and delays endosome maturation

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

doi: 10.1073/pnas.1806904115

Etf-2 has a GAP TBC-like dual catalytic finger motif that directly binds to RAB5-GTP but lacks RAB5 GAP activity. ( A , Upper ) Putative domain structures of Etf-2. Numbers indicate amino acid positions. Q, glutamine finger; R, arginine finger; the blue boxed domain is the putative T4SS signal. ( Lower ) Amino acid sequence alignment of Etf-2 with TBC domains of RABGAP5 and Shigella VirA that have RAB1 GAP activity. Etf-2 has the GAP TBC-like dual (Arg and Glu) catalytic finger motif (red) near its C terminus. ( B ) Direct interaction of full-length Etf-2 with RAB5-WT, -DN, and -CA was tested using the yeast two-hybrid assay. Twenty independent colonies were tested on SD/−Leu/−Trp double-dropout (DDO) and SD/−Leu/−Trp/−His/−Ade quadruple-dropout (QDO) agar plates. Growth on QDO plates indicated a specific interaction between Etf-2 and RAB5-WT or -CA. ( C ) MST assay with purified proteins demonstrating binding of RAB5A to Etf-2 ∆HY in a GTP-dependent manner. RAB5A was fluorescently labeled with Tris-nitrilotriacetic acid at fixed concentrations ranging from 5 to 200 nM and was preincubated with GTPγS or GDPβS; binding was assessed with unlabeled Etf-2 ∆HY from the low picomolar range to 100 µM. RAB5A binding to Etf-2 ∆HY was observed only in RAB5A-GTPγS samples. The determined K d value is shown. Data are shown as mean ± SD ( n = 4). ( D ) GAP activity of Etf-2 ∆HY . GST-RAB5 that had been affinity-purified on GST resin was preloaded with [α- 32 P]GTP followed by a GTP hydrolysis reaction in the absence or presence of RABGAP5 or Etf-2 ∆HY . Samples were taken at the indicated times and subjected to TLC followed by autoradiography and PhosphorImager analysis to visualize and quantify the radiolabeled hydrolysis product ( 32 P-GDP) from substrate ( 32 P-GTP) as indicated. The percentage values indicate the amount of GDP produced from GTP.
Figure Legend Snippet: Etf-2 has a GAP TBC-like dual catalytic finger motif that directly binds to RAB5-GTP but lacks RAB5 GAP activity. ( A , Upper ) Putative domain structures of Etf-2. Numbers indicate amino acid positions. Q, glutamine finger; R, arginine finger; the blue boxed domain is the putative T4SS signal. ( Lower ) Amino acid sequence alignment of Etf-2 with TBC domains of RABGAP5 and Shigella VirA that have RAB1 GAP activity. Etf-2 has the GAP TBC-like dual (Arg and Glu) catalytic finger motif (red) near its C terminus. ( B ) Direct interaction of full-length Etf-2 with RAB5-WT, -DN, and -CA was tested using the yeast two-hybrid assay. Twenty independent colonies were tested on SD/−Leu/−Trp double-dropout (DDO) and SD/−Leu/−Trp/−His/−Ade quadruple-dropout (QDO) agar plates. Growth on QDO plates indicated a specific interaction between Etf-2 and RAB5-WT or -CA. ( C ) MST assay with purified proteins demonstrating binding of RAB5A to Etf-2 ∆HY in a GTP-dependent manner. RAB5A was fluorescently labeled with Tris-nitrilotriacetic acid at fixed concentrations ranging from 5 to 200 nM and was preincubated with GTPγS or GDPβS; binding was assessed with unlabeled Etf-2 ∆HY from the low picomolar range to 100 µM. RAB5A binding to Etf-2 ∆HY was observed only in RAB5A-GTPγS samples. The determined K d value is shown. Data are shown as mean ± SD ( n = 4). ( D ) GAP activity of Etf-2 ∆HY . GST-RAB5 that had been affinity-purified on GST resin was preloaded with [α- 32 P]GTP followed by a GTP hydrolysis reaction in the absence or presence of RABGAP5 or Etf-2 ∆HY . Samples were taken at the indicated times and subjected to TLC followed by autoradiography and PhosphorImager analysis to visualize and quantify the radiolabeled hydrolysis product ( 32 P-GDP) from substrate ( 32 P-GTP) as indicated. The percentage values indicate the amount of GDP produced from GTP.

Techniques Used: Activity Assay, Sequencing, Y2H Assay, Microscale Thermophoresis, Purification, Binding Assay, Labeling, Affinity Purification, Thin Layer Chromatography, Autoradiography, Produced

2) Product Images from "Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation"

Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2018.02.011

α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.
Figure Legend Snippet: α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

Techniques Used: Selection, Incubation, Binding Assay, Amplification, Polymerase Chain Reaction, Purification, Magnetic Beads, Sequencing

3) Product Images from "STK25-induced inhibition of aerobic glycolysis via GOLPH3-mTOR pathway suppresses cell proliferation in colorectal cancer"

Article Title: STK25-induced inhibition of aerobic glycolysis via GOLPH3-mTOR pathway suppresses cell proliferation in colorectal cancer

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-018-0808-1

STK25 interacts with GOLPH3 and regulates its expression. a , b Exogenous STK25 interacts with GOLPH3. Cells were transfected with the indicated plasmids. Co-IP was performed using FLAG antibody to pull down FLAG-STK25 ( a ) or anti-Myc against Myc-GOLPH3 ( b ). Then, STK25 and GOLPH3 were detected with the indicated antibodies. c , d His-STK25 interacts directly with GST-GOLPH3 but not GST by in vitro GST pull-down and His-tag pull-down assays, respectively. e STK25 overexpression decreases GLOPH3 mRNA and protein levels in CRC cells. f STK25 knockdown increases GOLPH3 mRNA and protein levels in CRC cells
Figure Legend Snippet: STK25 interacts with GOLPH3 and regulates its expression. a , b Exogenous STK25 interacts with GOLPH3. Cells were transfected with the indicated plasmids. Co-IP was performed using FLAG antibody to pull down FLAG-STK25 ( a ) or anti-Myc against Myc-GOLPH3 ( b ). Then, STK25 and GOLPH3 were detected with the indicated antibodies. c , d His-STK25 interacts directly with GST-GOLPH3 but not GST by in vitro GST pull-down and His-tag pull-down assays, respectively. e STK25 overexpression decreases GLOPH3 mRNA and protein levels in CRC cells. f STK25 knockdown increases GOLPH3 mRNA and protein levels in CRC cells

Techniques Used: Expressing, Transfection, Co-Immunoprecipitation Assay, In Vitro, Over Expression

4) Product Images from "A phosphorylated transcription factor regulates sterol biosynthesis in Fusarium graminearum"

Article Title: A phosphorylated transcription factor regulates sterol biosynthesis in Fusarium graminearum

Journal: Nature Communications

doi: 10.1038/s41467-019-09145-6

A proposed model for the regulation of sterol biosynthesis mediated by FgSR in F. graminearum . Under the treatment with sterol biosynthesis inhibitors (SBIs), the HOG cascade FgSsk2-FgPbs2-FgHog1 is activated in F. graminearum . Subsequently, FgSR is strongly phosphorylated by activated FgHog1 and other kinase(s). The highly phosphorylated FgSR recruits the SWI/SNF complex to remodel chromatin, and subsequently induces the high transcription levels of sterol biosynthesis genes
Figure Legend Snippet: A proposed model for the regulation of sterol biosynthesis mediated by FgSR in F. graminearum . Under the treatment with sterol biosynthesis inhibitors (SBIs), the HOG cascade FgSsk2-FgPbs2-FgHog1 is activated in F. graminearum . Subsequently, FgSR is strongly phosphorylated by activated FgHog1 and other kinase(s). The highly phosphorylated FgSR recruits the SWI/SNF complex to remodel chromatin, and subsequently induces the high transcription levels of sterol biosynthesis genes

Techniques Used:

Phosphorylation of FgSR mediated by FgHog1 regulates the expression of FgCYP51A . a Deletion of kinase in the HOG cascade or mutations at the predicted phosphorylation sites within FgSR led to increased sensitivity to tebuconazole. b Comparisons of expression level of FgCYP51A among the above strains after the treatment with 2.5 μg ml –1 tebuconazole for 6 h. The expression level of FgCYP51A in the wild type without treatment was referred to 1. Data presented are the mean ± s.d. ( n = 3). Bars followed by the same letter are not significantly different according to a LSD test at P = 0.01. c The phosphorylation of FgHog1 was increased by the treatment with 2.5 μg ml –1 tebuconazole. PH-1 was treated with tebuconazole for 0, 0.5, 1 and 2 h after incubated in YEPD for 36 h. d Localization of FgHog1-GFP with 2.5 μg ml –1 tebuconazole treatment for 0 and 2 h. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). DIC, differential interference contrast. Bar = 10 μm. e Loss of FgSsk2, FgPbs2 or FgHog1 decreased the phosphorylation of FgSR with or without tebuconazole treatment. The dephosphorylated and phosphorylated FgSR are indicated with red and blue arrows, respectively. f FgHog1 interacts with FgSR in the co-immunoprecipitation (Co-IP) assay and the interaction is dependent on FgSsk2 and enhanced by tebuconazole treatment. The strains were treated with (+) or without (−) tebuconazole for 2 h after incubated in YEPD for 36 h. g FgHog1 interacts with FgSR in the nucleus under the treatment with 2.5 μg ml –1 tebuconazole, and the interaction is dependent on the FgSsk2 by bimolecular fluorescence complementation (BiFC) assays. Bar = 10 μm. h The interaction of FgHog1 and FgSR assayed using GST pull-downs. Proteins were also detected by staining with Coomassie brilliant blue (CBB). i Phosphorylation of FgSR by Hog1 in vitro. Phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography (upper panel). GST-FgHog1 and His-FgSR were also detected by staining with CBB (lower panel). j Position of the five predicted phosphorylated amino acid residues in FgSR
Figure Legend Snippet: Phosphorylation of FgSR mediated by FgHog1 regulates the expression of FgCYP51A . a Deletion of kinase in the HOG cascade or mutations at the predicted phosphorylation sites within FgSR led to increased sensitivity to tebuconazole. b Comparisons of expression level of FgCYP51A among the above strains after the treatment with 2.5 μg ml –1 tebuconazole for 6 h. The expression level of FgCYP51A in the wild type without treatment was referred to 1. Data presented are the mean ± s.d. ( n = 3). Bars followed by the same letter are not significantly different according to a LSD test at P = 0.01. c The phosphorylation of FgHog1 was increased by the treatment with 2.5 μg ml –1 tebuconazole. PH-1 was treated with tebuconazole for 0, 0.5, 1 and 2 h after incubated in YEPD for 36 h. d Localization of FgHog1-GFP with 2.5 μg ml –1 tebuconazole treatment for 0 and 2 h. Nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). DIC, differential interference contrast. Bar = 10 μm. e Loss of FgSsk2, FgPbs2 or FgHog1 decreased the phosphorylation of FgSR with or without tebuconazole treatment. The dephosphorylated and phosphorylated FgSR are indicated with red and blue arrows, respectively. f FgHog1 interacts with FgSR in the co-immunoprecipitation (Co-IP) assay and the interaction is dependent on FgSsk2 and enhanced by tebuconazole treatment. The strains were treated with (+) or without (−) tebuconazole for 2 h after incubated in YEPD for 36 h. g FgHog1 interacts with FgSR in the nucleus under the treatment with 2.5 μg ml –1 tebuconazole, and the interaction is dependent on the FgSsk2 by bimolecular fluorescence complementation (BiFC) assays. Bar = 10 μm. h The interaction of FgHog1 and FgSR assayed using GST pull-downs. Proteins were also detected by staining with Coomassie brilliant blue (CBB). i Phosphorylation of FgSR by Hog1 in vitro. Phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography (upper panel). GST-FgHog1 and His-FgSR were also detected by staining with CBB (lower panel). j Position of the five predicted phosphorylated amino acid residues in FgSR

Techniques Used: Expressing, Incubation, Staining, Co-Immunoprecipitation Assay, Fluorescence, Bimolecular Fluorescence Complementation Assay, In Vitro, SDS Page, Autoradiography

5) Product Images from "PfMSA180 is a novel Plasmodium falciparum vaccine antigen that interacts with human erythrocyte integrin associated protein (CD47)"

Article Title: PfMSA180 is a novel Plasmodium falciparum vaccine antigen that interacts with human erythrocyte integrin associated protein (CD47)

Journal: Scientific Reports

doi: 10.1038/s41598-019-42366-9

PfMSA180 (PF3D7_1014100) exists as a 170 kDa protein in parasites. ( A ) Schematic representation of full-length PfMSA180 and recombinant protein fragments (Tr 1–5) PfMSA180 consists of 1455 aa with a calculated molecular mass (MW) of 173.3 kDa. The protein has a predicted signal peptide (SP; 1 to 22 aa; shown in green). Recombinant PfMSA180 truncates were expressed as N-terminal GST-tagged proteins by the wheat germ cell-free system (WGCFS). Tr1, residues E 22 -S 263 (expected MW 29 kDa); Tr2, A 264 -D 501 (expected MW 27.9 kDa); Tr3, I 508 -P 723 (expected MW 52.8 kDa, including the GST tag); Tr4, A 805 -P 1093 (expected MW 34 0.4 kDa); and Tr5, L 1193 -P 1455 (expected MW 58.5 kDa including the GST tag). ( B ) Recombinant PfMSA180 truncates are shown stained with Coomassie brilliant blue (CBB) following purification on a glutathione-Sepharose 4B column and resolution by 12.5% SDS-PAGE under reducing conditions. Arrowheads indicate molecular masses predicted from amino acid sequences of the corresponding recombinant PfMSA180 truncates (original data is included in the Supplementary Data File 1 ). ( C ) Reactivity of rabbit anti-PfMSA180 antibodies to native PfMSA180. Schizont-rich parasite pellets were solubilized with NP40 and PfMSA180 immunoprecipitated with the indicated mouse antibodies. Mouse anti-HisGST antibody was used as a negative control. Immunoprecipitated full length PfMSA180 was detected using rabbit antibody to each of the truncates; indicated using an arrowhead. The additional bands observed in the membrane fraction at 120, 80, and 45 kDa, were likely products of protein SUB1-mediated proteolysis of full-length protein. Antibodies to truncate 3 did not immunoprecipitate protein (original data is included in the Supplementary Data File 1 ).
Figure Legend Snippet: PfMSA180 (PF3D7_1014100) exists as a 170 kDa protein in parasites. ( A ) Schematic representation of full-length PfMSA180 and recombinant protein fragments (Tr 1–5) PfMSA180 consists of 1455 aa with a calculated molecular mass (MW) of 173.3 kDa. The protein has a predicted signal peptide (SP; 1 to 22 aa; shown in green). Recombinant PfMSA180 truncates were expressed as N-terminal GST-tagged proteins by the wheat germ cell-free system (WGCFS). Tr1, residues E 22 -S 263 (expected MW 29 kDa); Tr2, A 264 -D 501 (expected MW 27.9 kDa); Tr3, I 508 -P 723 (expected MW 52.8 kDa, including the GST tag); Tr4, A 805 -P 1093 (expected MW 34 0.4 kDa); and Tr5, L 1193 -P 1455 (expected MW 58.5 kDa including the GST tag). ( B ) Recombinant PfMSA180 truncates are shown stained with Coomassie brilliant blue (CBB) following purification on a glutathione-Sepharose 4B column and resolution by 12.5% SDS-PAGE under reducing conditions. Arrowheads indicate molecular masses predicted from amino acid sequences of the corresponding recombinant PfMSA180 truncates (original data is included in the Supplementary Data File 1 ). ( C ) Reactivity of rabbit anti-PfMSA180 antibodies to native PfMSA180. Schizont-rich parasite pellets were solubilized with NP40 and PfMSA180 immunoprecipitated with the indicated mouse antibodies. Mouse anti-HisGST antibody was used as a negative control. Immunoprecipitated full length PfMSA180 was detected using rabbit antibody to each of the truncates; indicated using an arrowhead. The additional bands observed in the membrane fraction at 120, 80, and 45 kDa, were likely products of protein SUB1-mediated proteolysis of full-length protein. Antibodies to truncate 3 did not immunoprecipitate protein (original data is included in the Supplementary Data File 1 ).

Techniques Used: Recombinant, Staining, Purification, SDS Page, Immunoprecipitation, Negative Control

PfMSA180 is expressed in schizonts and localizes on the merozoite surface. ( A ) A time course immunoblot analysis of 3D7 parasite extracts shows that PfMSA180 is synthesized late in the intraerythrocytic cycle. The protein extracts prepared from Percoll-sorbitol-synchronized parasites were electrophoresed on SDS-12.5% PAGE and probed with rabbit polyclonal anti-PfMSA180 Tr1 antibodies. Hours post-invasion (hpi) are indicated for each lane. Anti-PfHSP70 monoclonal antibody was used as a quantitative parasite protein marker, anti-human spectrin α I rabbit antibody (Santa Cruz Biotechnology, Dallas, TX) indicating the number of loaded erythrocytes 49 , and anti-AMA1 antibody was used as a mature schizont stage marker (original data is included in the Supplementary Data File 1 ). ( B ) IFA analysis of PfMSA180 with MTIP (myosin A tail domain interacting protein). Free merozoites were processed with (+) or without (−) permeabilization using 0.1% Triton X-100 (TrX-100). The merozoites were stained with anti-PfMSA180-Tr1 antibody (upper panels, green color) or anti-PfMSA180-Tr4 (lower panels, green color) and co-stained with anti-MTIP antibody (red color). The leftmost panels show pictures of differential interference contrast (DIC). The rightmost panels show merged pictures with DAPI, showing localization of parasite’s nucleus. ( C ) IFA analysis of PfMSA180 with AMA1 and MSP1-19. Free merozoites were permeabilized with 0.1% Triton X-100. The merozoites were stained with anti-PfMSA180-Tr1 antibody (upper panels, green color) or anti-PfMSA180-Tr4 (lower panels, green color) and co-stained with anti-AMA1 antibody or anti-MSP1-19 antibody (red color). The leftmost panels show pictures of differential interference contrast (DIC). The rightmost panels show merged pictures with DAPI, showing localization of parasite’s nucleus. ( D ) Fractionation of PfMSA180 due to solubility. Schizont-rich 3D7 parasites were disrupted by sonication and fractionated with ultracentrifugation. The resulting soluble and membrane fractions were applied to SDS-PAGE. Immunoblotting analysis with anti-Tr1 antibody detected PfMSA180 in the membrane fraction. MSP3, a secreted protein was detected in both membrane and soluble fractions. AMA1, an integral membrane protein, and MSP1-19, a peripheral GPI-anchored membrane protein, were detected in the membrane fraction. TL: total schizont-rich parasite lysate; S: soluble fractions; M: membrane fraction. Original data is included in the Supplementary Data File 1 .
Figure Legend Snippet: PfMSA180 is expressed in schizonts and localizes on the merozoite surface. ( A ) A time course immunoblot analysis of 3D7 parasite extracts shows that PfMSA180 is synthesized late in the intraerythrocytic cycle. The protein extracts prepared from Percoll-sorbitol-synchronized parasites were electrophoresed on SDS-12.5% PAGE and probed with rabbit polyclonal anti-PfMSA180 Tr1 antibodies. Hours post-invasion (hpi) are indicated for each lane. Anti-PfHSP70 monoclonal antibody was used as a quantitative parasite protein marker, anti-human spectrin α I rabbit antibody (Santa Cruz Biotechnology, Dallas, TX) indicating the number of loaded erythrocytes 49 , and anti-AMA1 antibody was used as a mature schizont stage marker (original data is included in the Supplementary Data File 1 ). ( B ) IFA analysis of PfMSA180 with MTIP (myosin A tail domain interacting protein). Free merozoites were processed with (+) or without (−) permeabilization using 0.1% Triton X-100 (TrX-100). The merozoites were stained with anti-PfMSA180-Tr1 antibody (upper panels, green color) or anti-PfMSA180-Tr4 (lower panels, green color) and co-stained with anti-MTIP antibody (red color). The leftmost panels show pictures of differential interference contrast (DIC). The rightmost panels show merged pictures with DAPI, showing localization of parasite’s nucleus. ( C ) IFA analysis of PfMSA180 with AMA1 and MSP1-19. Free merozoites were permeabilized with 0.1% Triton X-100. The merozoites were stained with anti-PfMSA180-Tr1 antibody (upper panels, green color) or anti-PfMSA180-Tr4 (lower panels, green color) and co-stained with anti-AMA1 antibody or anti-MSP1-19 antibody (red color). The leftmost panels show pictures of differential interference contrast (DIC). The rightmost panels show merged pictures with DAPI, showing localization of parasite’s nucleus. ( D ) Fractionation of PfMSA180 due to solubility. Schizont-rich 3D7 parasites were disrupted by sonication and fractionated with ultracentrifugation. The resulting soluble and membrane fractions were applied to SDS-PAGE. Immunoblotting analysis with anti-Tr1 antibody detected PfMSA180 in the membrane fraction. MSP3, a secreted protein was detected in both membrane and soluble fractions. AMA1, an integral membrane protein, and MSP1-19, a peripheral GPI-anchored membrane protein, were detected in the membrane fraction. TL: total schizont-rich parasite lysate; S: soluble fractions; M: membrane fraction. Original data is included in the Supplementary Data File 1 .

Techniques Used: Synthesized, Polyacrylamide Gel Electrophoresis, Marker, Immunofluorescence, Staining, Fractionation, Solubility, Sonication, SDS Page

6) Product Images from "BCAS2 Enhances Carcinogenic Effects of Estrogen Receptor Alpha in Breast Cancer Cells"

Article Title: BCAS2 Enhances Carcinogenic Effects of Estrogen Receptor Alpha in Breast Cancer Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20040966

BCAS2 interacts with ERα in vivo and in vitro. ( A ) Structure of ERα and its N and C domains used for Glutathione sepharose affinity matrix assays. NTD, amino terminal domain; DBD, DNA binding domain; HR, hinge region; LBD, ligand binding domain. ( B ) GST pull-down assays of biotin labeled in vitro translated BCAS2 with GST alone, GST-ERα-Full (full-length aa 1-595), GST-ERα-N (aa 1-180) GST-ERα-C (aa 264-595). Western blot analysis was carried out using anti-biotin or anti-GST antibodies. Binding was assayed in the presence (+) or absence (−) of 100 nM E2. ( C ) Coimmunoprecipitation of ERα and BCAS2. COS7 cells were transfected with plasmids expressing ERα and BCAS2 in the presence (+) or absence (−) of 10 nM E2. Immunoprecipitation of whole cell protein extracts was carried out with antibodies against BCAS2 or ERα and IgG as negative control. Shown are immunoblots probed with antibodies against both proteins. ( D ) Coimmunoprecipitation assays in MCF7 cells in the presence of ethanol (OH), E 2 (10 nM) or TAM (100 nM) showing interaction in the presence or absence of ligand. Full blots can be found in supplementary Figure S1 .
Figure Legend Snippet: BCAS2 interacts with ERα in vivo and in vitro. ( A ) Structure of ERα and its N and C domains used for Glutathione sepharose affinity matrix assays. NTD, amino terminal domain; DBD, DNA binding domain; HR, hinge region; LBD, ligand binding domain. ( B ) GST pull-down assays of biotin labeled in vitro translated BCAS2 with GST alone, GST-ERα-Full (full-length aa 1-595), GST-ERα-N (aa 1-180) GST-ERα-C (aa 264-595). Western blot analysis was carried out using anti-biotin or anti-GST antibodies. Binding was assayed in the presence (+) or absence (−) of 100 nM E2. ( C ) Coimmunoprecipitation of ERα and BCAS2. COS7 cells were transfected with plasmids expressing ERα and BCAS2 in the presence (+) or absence (−) of 10 nM E2. Immunoprecipitation of whole cell protein extracts was carried out with antibodies against BCAS2 or ERα and IgG as negative control. Shown are immunoblots probed with antibodies against both proteins. ( D ) Coimmunoprecipitation assays in MCF7 cells in the presence of ethanol (OH), E 2 (10 nM) or TAM (100 nM) showing interaction in the presence or absence of ligand. Full blots can be found in supplementary Figure S1 .

Techniques Used: In Vivo, In Vitro, Binding Assay, Ligand Binding Assay, Labeling, Western Blot, Transfection, Expressing, Immunoprecipitation, Negative Control

7) Product Images from "Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4"

Article Title: Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4

Journal: Scientific Reports

doi: 10.1038/s41598-018-38134-w

Introduction of mutations in predicted Can f 6 epitopes. ( A ) Highly conserved region among Can f 6, Fel d 4, and Equ c 1—but not among other representative lipocalin allergens (indicated by a green bar above the sequences)—was predicted to contain IgE epitope(s) involved in cross-reactivity. Three sites composed of three successive amino acids containing charged residues were substituted with triple alanine, and were designated as rCan f 6-mu-1, mu-2, and mu-3. ( B ) Schematic representation of the mutation sites (shown in red) in a ribbon diagram (left) and surface model (right) of rCan f 6.
Figure Legend Snippet: Introduction of mutations in predicted Can f 6 epitopes. ( A ) Highly conserved region among Can f 6, Fel d 4, and Equ c 1—but not among other representative lipocalin allergens (indicated by a green bar above the sequences)—was predicted to contain IgE epitope(s) involved in cross-reactivity. Three sites composed of three successive amino acids containing charged residues were substituted with triple alanine, and were designated as rCan f 6-mu-1, mu-2, and mu-3. ( B ) Schematic representation of the mutation sites (shown in red) in a ribbon diagram (left) and surface model (right) of rCan f 6.

Techniques Used: Mutagenesis

X-ray crystal structure of Can f 6 with characteristics typical of lipocalin-like proteins. ( A ) Tertiary structure of rCan f 6 (chain A) represented in a ribbon diagram. A β-barrel structure composed of 8 β-strands (indicated by A–H) is shown in green, while an α-helix and 3 10 -helix structures are indicated in orange. Yellow sticks represent an intramolecular disulfide bond. ( B ) Positions of typical secondary structures in rCan f 6 (chain A) are represented along with its amino acid sequence.
Figure Legend Snippet: X-ray crystal structure of Can f 6 with characteristics typical of lipocalin-like proteins. ( A ) Tertiary structure of rCan f 6 (chain A) represented in a ribbon diagram. A β-barrel structure composed of 8 β-strands (indicated by A–H) is shown in green, while an α-helix and 3 10 -helix structures are indicated in orange. Yellow sticks represent an intramolecular disulfide bond. ( B ) Positions of typical secondary structures in rCan f 6 (chain A) are represented along with its amino acid sequence.

Techniques Used: Sequencing

Effect of predicted Can f 6 epitope mutations on IgE reactivity. ( A ) Relative IgE reactivity to the mutated rCan f 6 proteins compared with rCan f 6 was evaluated by ELISA. Can f 6-reactive sera from 18 patients were subjected to this assay. Lines in individual columns denote the median. ( B ) Inhibition ELISA to assess specific IgE binding to immobilized rCan f 6 in the presense of wild-type and mutated rCan f 6 proteins as competitors. Sera from patients 16, 25 and 35 were used for this assay. ( C ) IgE-western blotting of Can f 6. rCan f 6 and mutated rCan f 6 proteins were subjected to SDS-PAGE and then transferred to PVDF membranes. The blots were treated with the serum from the 3 patients (upper panels). The blots were also stained with Ponceau S to verify proper protein transfer (lower panels).
Figure Legend Snippet: Effect of predicted Can f 6 epitope mutations on IgE reactivity. ( A ) Relative IgE reactivity to the mutated rCan f 6 proteins compared with rCan f 6 was evaluated by ELISA. Can f 6-reactive sera from 18 patients were subjected to this assay. Lines in individual columns denote the median. ( B ) Inhibition ELISA to assess specific IgE binding to immobilized rCan f 6 in the presense of wild-type and mutated rCan f 6 proteins as competitors. Sera from patients 16, 25 and 35 were used for this assay. ( C ) IgE-western blotting of Can f 6. rCan f 6 and mutated rCan f 6 proteins were subjected to SDS-PAGE and then transferred to PVDF membranes. The blots were treated with the serum from the 3 patients (upper panels). The blots were also stained with Ponceau S to verify proper protein transfer (lower panels).

Techniques Used: Enzyme-linked Immunosorbent Assay, Inhibition, Binding Assay, Western Blot, SDS Page, Staining

Purification of rCan f 6. ( A ) Gel filtration chromatogram of the purified rCan f 6. ( B , C ) SDS-PAGE profiles of rCan f 6. Purified recombinant protein (3 µg/lane) was electrophoretically separated under ( B ) reducing or ( C ) non-reducing conditions and then stained with Coomassie Brilliant blue. ( D ) Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectra of rCan f 6. Mass spectrometry of the purified recombinant protein was carried out in the linear mode using sinapinic acid as a matrix. The  m/z  value of the main peak (20337.47) corresponds to the deduced molecular mass of the recombinant protein. The sub-peak ( m/z  20550) is considered to be derived from rCan f 6 complexed with sinapinic acid. ( E ) Distribution states of rCan f 6 analysed by AUC-SV. The molecular mass of rCan f 6 was calculated as 19.9 kDa.
Figure Legend Snippet: Purification of rCan f 6. ( A ) Gel filtration chromatogram of the purified rCan f 6. ( B , C ) SDS-PAGE profiles of rCan f 6. Purified recombinant protein (3 µg/lane) was electrophoretically separated under ( B ) reducing or ( C ) non-reducing conditions and then stained with Coomassie Brilliant blue. ( D ) Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectra of rCan f 6. Mass spectrometry of the purified recombinant protein was carried out in the linear mode using sinapinic acid as a matrix. The m/z value of the main peak (20337.47) corresponds to the deduced molecular mass of the recombinant protein. The sub-peak ( m/z 20550) is considered to be derived from rCan f 6 complexed with sinapinic acid. ( E ) Distribution states of rCan f 6 analysed by AUC-SV. The molecular mass of rCan f 6 was calculated as 19.9 kDa.

Techniques Used: Purification, Filtration, SDS Page, Recombinant, Staining, Mass Spectrometry, Derivative Assay

Analysis of IgE cross-reactivity between Can f 6 and Fel d 4. Inhibition ELISA to assess specific IgE binding to immobilized rFel d 4 in the presence of rFel d 4, wild-type and mutated rCan f 6 proteins as competitors. Sera from patients 16, 25 and 35 were used for this assay.
Figure Legend Snippet: Analysis of IgE cross-reactivity between Can f 6 and Fel d 4. Inhibition ELISA to assess specific IgE binding to immobilized rFel d 4 in the presence of rFel d 4, wild-type and mutated rCan f 6 proteins as competitors. Sera from patients 16, 25 and 35 were used for this assay.

Techniques Used: Inhibition, Enzyme-linked Immunosorbent Assay, Binding Assay

8) Product Images from "The LIM protein Ajuba recruits DBC1 and CBP/p300 to acetylate ERα and enhances ERα target gene expression in breast cancer cells"

Article Title: The LIM protein Ajuba recruits DBC1 and CBP/p300 to acetylate ERα and enhances ERα target gene expression in breast cancer cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky1306

Ajuba interacts with ERα independent of the conserved NR boxes. ( A ) The three conserved NR-boxes in Ajuba were shown on the upper panel. Plasmids were transiently transfected into 293T cells and the co-IP assay was carried out with Myc antibody. * IgG-heavy chain. ( B ) Flag-ERα and Myc-Ajuba plasmids were transfected into 293T cells and co-IP assay was performed by using Flag-M2-beads. ( C ) The preLIM and LIM regions of Ajuba were illustrated on the upper panel. The full length or truncations of Ajuba were transiently co-transfected into 293T cells together with Flag-ERα plasmid. The co-IP assay was performed by using Flag antibody. ( D ) The functional domains of ERα were shown on the upper panel. The interaction between full-length or truncations of ERα with Ajuba in 293T cells was detected by co-IP assay and western blotting. The relative amount of co-eluted Myc-Ajuba was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled. ( E ) The plasmids encoding Ajuba, LIMD1, Zyxin, Wtip and Lpp were respectively co-transfected with Flag-ERα plasmid into 293T cells and co-IP assay was performed. ( F ) The plasmids encoding Flag-ERα and Myc-Ajuba were transfected into 293T cells, and the resulting cells were cultured in phenol-red free media containing 5% charcoal stripped FBS for 2 days and then treated with E2 (20 or 100 nM) or ethanol for 12 h. The co-IP assay was performed by using Flag-M2 beads. The relative amount of immunoprecipitated Myc-Ajuba was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled. ( G ) GST-ERα and His-Ajuba proteins were expressed in E. coli BL21, and GST-pulldown assay was performed in the presence of E2 (100 nM) or ethanol. The relative amount of pulled-down His-Ajuba was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled. ( H ) T47D cells treated with 100 nM E2 or ethanol for 12 h were harvested and co-IP assay was performed by using ERα antibody or IgG control. The relative amount of immunoprecipitated Ajuba was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled.
Figure Legend Snippet: Ajuba interacts with ERα independent of the conserved NR boxes. ( A ) The three conserved NR-boxes in Ajuba were shown on the upper panel. Plasmids were transiently transfected into 293T cells and the co-IP assay was carried out with Myc antibody. * IgG-heavy chain. ( B ) Flag-ERα and Myc-Ajuba plasmids were transfected into 293T cells and co-IP assay was performed by using Flag-M2-beads. ( C ) The preLIM and LIM regions of Ajuba were illustrated on the upper panel. The full length or truncations of Ajuba were transiently co-transfected into 293T cells together with Flag-ERα plasmid. The co-IP assay was performed by using Flag antibody. ( D ) The functional domains of ERα were shown on the upper panel. The interaction between full-length or truncations of ERα with Ajuba in 293T cells was detected by co-IP assay and western blotting. The relative amount of co-eluted Myc-Ajuba was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled. ( E ) The plasmids encoding Ajuba, LIMD1, Zyxin, Wtip and Lpp were respectively co-transfected with Flag-ERα plasmid into 293T cells and co-IP assay was performed. ( F ) The plasmids encoding Flag-ERα and Myc-Ajuba were transfected into 293T cells, and the resulting cells were cultured in phenol-red free media containing 5% charcoal stripped FBS for 2 days and then treated with E2 (20 or 100 nM) or ethanol for 12 h. The co-IP assay was performed by using Flag-M2 beads. The relative amount of immunoprecipitated Myc-Ajuba was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled. ( G ) GST-ERα and His-Ajuba proteins were expressed in E. coli BL21, and GST-pulldown assay was performed in the presence of E2 (100 nM) or ethanol. The relative amount of pulled-down His-Ajuba was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled. ( H ) T47D cells treated with 100 nM E2 or ethanol for 12 h were harvested and co-IP assay was performed by using ERα antibody or IgG control. The relative amount of immunoprecipitated Ajuba was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled.

Techniques Used: Transfection, Co-Immunoprecipitation Assay, Plasmid Preparation, Functional Assay, Western Blot, Labeling, Cell Culture, Immunoprecipitation, GST Pulldown Assay

Ajuba recruits DBC1 to enhance ERα transcriptional activity. (A, B) plasmids encoding DBC1 and Ajuba were transfected into 293T cells and co-IP assay was performed by using Myc antibody ( A ) or Flag-M2-beads ( B ). ( C ) The endogenous interaction between DBC1 and Ajuba was detected in T47D cells by co-IP assay. ( D ) GST-DBC1 and His-Ajuba was respectively expressed in E. coli BL21, and in vitro binding assay was performed. ( E ) The plasmids of full-length and truncations of Ajuba protein were co-transfected into 293T cells with DBC1 plasmids and co-IP assay was carried out. ( F ) Increasing amount of plasmids encoding Ajuba were co-expressed along with DBC1 and ERα in 293T cells and co-IP assay showed that Ajuba enhanced the interaction between DBC1 and ERα (the relative amount of immunoprecipitated HA-DBC1 was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled). ( G ) TFF1 promoter driven luciferase reporter assay was performed in 293T cells and the transfected cells were treated with 20 nM E2 before detecting luciferase activity and the value was normalized to β-gal (three repeats, * means P
Figure Legend Snippet: Ajuba recruits DBC1 to enhance ERα transcriptional activity. (A, B) plasmids encoding DBC1 and Ajuba were transfected into 293T cells and co-IP assay was performed by using Myc antibody ( A ) or Flag-M2-beads ( B ). ( C ) The endogenous interaction between DBC1 and Ajuba was detected in T47D cells by co-IP assay. ( D ) GST-DBC1 and His-Ajuba was respectively expressed in E. coli BL21, and in vitro binding assay was performed. ( E ) The plasmids of full-length and truncations of Ajuba protein were co-transfected into 293T cells with DBC1 plasmids and co-IP assay was carried out. ( F ) Increasing amount of plasmids encoding Ajuba were co-expressed along with DBC1 and ERα in 293T cells and co-IP assay showed that Ajuba enhanced the interaction between DBC1 and ERα (the relative amount of immunoprecipitated HA-DBC1 was semi-quantified by grayscale analysis and the mean values of the three repeats were labeled). ( G ) TFF1 promoter driven luciferase reporter assay was performed in 293T cells and the transfected cells were treated with 20 nM E2 before detecting luciferase activity and the value was normalized to β-gal (three repeats, * means P

Techniques Used: Activity Assay, Transfection, Co-Immunoprecipitation Assay, In Vitro, Binding Assay, Immunoprecipitation, Labeling, Luciferase, Reporter Assay

9) Product Images from "Non-catalytic signaling by pseudokinase ILK for regulating cell adhesion"

Article Title: Non-catalytic signaling by pseudokinase ILK for regulating cell adhesion

Journal: Nature Communications

doi: 10.1038/s41467-018-06906-7

ILK L207W mutation disrupts the F-actin bundling. a (Top left) Structural comparison of the ILK KLD bound to α-Parvin CH2 and (colored in gray; PDB ID 3KMW) and its comparison with the ILK mutant form (colored in blue). The superposition of the mutant ILK KLD (267 aligned Cα atoms) to the ATP-bound (PDB ID 3KMW) and -free (PDB ID 3KMU) forms shows overall similarities with root-mean-square deviations of 0.58 Å and 0.47 Å, respectively. A small conformational change is observed in the ATP-binding site of the mutant ILK KLD likely due to mutation or distinct crystal packing. (Top right) Close-up view of the ATP-binding sites of the ILK KLD between ATP-bound wild type (gray) and deficient mutant (blue). Mg, ATP, L207 in the wild type, and W207 in the mutant ILK KLDs are highlighted in ball and stick models. (Bottom) Close-up stereo view of the loss-of-ATP-binding mutation site in the ILK KLD. The 2Fo-Fc electron density map contoured at 1 σ is shown in gray mesh. The Fo–Fc omit map calculated from the mutant structure without the residue (W207), contoured at 3.5 σ, is overlaid (red mesh). Selected residues in the ATP-binding site are labeled. b Representative microscopic image showing that IPP L207W impaired F-actin bundle formation (no larger bundles) as compared with the WT IPP in ( c ). Selected microscopic image showing F-actin bundles in the presence of WT IPP. Bar = 100 μm. d Quantitative comparison of the F-actin bundle sizes of the randomly selected 20 slides showing the mutation dramatically reduced the F-actin bundle sizes (red squares) as compared with those induced by WT IPP (blue triangles)
Figure Legend Snippet: ILK L207W mutation disrupts the F-actin bundling. a (Top left) Structural comparison of the ILK KLD bound to α-Parvin CH2 and (colored in gray; PDB ID 3KMW) and its comparison with the ILK mutant form (colored in blue). The superposition of the mutant ILK KLD (267 aligned Cα atoms) to the ATP-bound (PDB ID 3KMW) and -free (PDB ID 3KMU) forms shows overall similarities with root-mean-square deviations of 0.58 Å and 0.47 Å, respectively. A small conformational change is observed in the ATP-binding site of the mutant ILK KLD likely due to mutation or distinct crystal packing. (Top right) Close-up view of the ATP-binding sites of the ILK KLD between ATP-bound wild type (gray) and deficient mutant (blue). Mg, ATP, L207 in the wild type, and W207 in the mutant ILK KLDs are highlighted in ball and stick models. (Bottom) Close-up stereo view of the loss-of-ATP-binding mutation site in the ILK KLD. The 2Fo-Fc electron density map contoured at 1 σ is shown in gray mesh. The Fo–Fc omit map calculated from the mutant structure without the residue (W207), contoured at 3.5 σ, is overlaid (red mesh). Selected residues in the ATP-binding site are labeled. b Representative microscopic image showing that IPP L207W impaired F-actin bundle formation (no larger bundles) as compared with the WT IPP in ( c ). Selected microscopic image showing F-actin bundles in the presence of WT IPP. Bar = 100 μm. d Quantitative comparison of the F-actin bundle sizes of the randomly selected 20 slides showing the mutation dramatically reduced the F-actin bundle sizes (red squares) as compared with those induced by WT IPP (blue triangles)

Techniques Used: Mutagenesis, Binding Assay, Labeling

IPP interaction with F-actin. a Schematic organization of IPP based on structural data. ILK binds to PINCH LIM1 via its ankyrin domain and α-Parvin CH2 via its pseudokinase domain, respectively. The Wiscott–Aldrich syndrome protein (WASP) homology domain (WH2) motifs are highlighted in PINCH and α-Parvin. b A representative gel filtration profile of the purified IPP complex by Superose 6 10/300 GL size exclusion chromatography column (GE healthcare). The eluted peak is overlaid with an elution curve of standard molecular weight proteins (dot lines). c Co-sedimentation of IPP at dose-dependent amounts in the presence/absence of F-actin. The F-actin was incubated at 2.3 μM constant concentration with increasing concentrations of each test sample in 5% glycerol containing protein buffer. Representative gels with Coomassie stain are shown. M marker proteins, S supernatant, P pellets
Figure Legend Snippet: IPP interaction with F-actin. a Schematic organization of IPP based on structural data. ILK binds to PINCH LIM1 via its ankyrin domain and α-Parvin CH2 via its pseudokinase domain, respectively. The Wiscott–Aldrich syndrome protein (WASP) homology domain (WH2) motifs are highlighted in PINCH and α-Parvin. b A representative gel filtration profile of the purified IPP complex by Superose 6 10/300 GL size exclusion chromatography column (GE healthcare). The eluted peak is overlaid with an elution curve of standard molecular weight proteins (dot lines). c Co-sedimentation of IPP at dose-dependent amounts in the presence/absence of F-actin. The F-actin was incubated at 2.3 μM constant concentration with increasing concentrations of each test sample in 5% glycerol containing protein buffer. Representative gels with Coomassie stain are shown. M marker proteins, S supernatant, P pellets

Techniques Used: Filtration, Purification, Size-exclusion Chromatography, Molecular Weight, Sedimentation, Incubation, Concentration Assay, Staining, Marker

Identification of actin-binding WH2 motifs in PINCH-1 and α-Parvin. a Sequence alignment of PINCH-1 C-terminal tail and α--terminus with representative WH2 motifs found in other proteins showing the presence of distinct WH2 motifs in PINCH and Parvin. b 0.1 mM 1 H- 15 N HSQC of α-Parvin-N in the absence and presence of 0.2 mM AP-actin showing that α-Parvin-N has potent binding to G-actin (left panel). Mutation of putative G-actin-binding residues L37A/R39A/R40A/K41A/K42A (α-Parvin 5 A) drastically reduces actin binding (right panel). c Co-sedimentation assay showing α-Parvin-N binds to F-actin potently. +, ++, +++ correspond to the concentration of α-Parvin-N at 7.7 μM, 23.0 μM, and 76.7 μM, respectively
Figure Legend Snippet: Identification of actin-binding WH2 motifs in PINCH-1 and α-Parvin. a Sequence alignment of PINCH-1 C-terminal tail and α--terminus with representative WH2 motifs found in other proteins showing the presence of distinct WH2 motifs in PINCH and Parvin. b 0.1 mM 1 H- 15 N HSQC of α-Parvin-N in the absence and presence of 0.2 mM AP-actin showing that α-Parvin-N has potent binding to G-actin (left panel). Mutation of putative G-actin-binding residues L37A/R39A/R40A/K41A/K42A (α-Parvin 5 A) drastically reduces actin binding (right panel). c Co-sedimentation assay showing α-Parvin-N binds to F-actin potently. +, ++, +++ correspond to the concentration of α-Parvin-N at 7.7 μM, 23.0 μM, and 76.7 μM, respectively

Techniques Used: Binding Assay, Sequencing, Mutagenesis, Sedimentation, Concentration Assay

10) Product Images from "The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation"

Article Title: The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.9.3577-3587.2004

Effect of PABP and Apc5 on translation in vitro. (A) Krebs-2 cell-free translation reaction mixtures that had been pretreated with GST-Paip2 for PABP depletion or with GST as a control were programmed with 100 ng of the indicated transcripts at 37°C for 90 min followed by measurements of firefly luciferase activity. Immunoblot analysis of the translation extracts using antibodies specific for PABP is shown at the top: lane 1, untreated; lane 2, treated with GST-Paip2; lane 3, treated with GST. For each transcript, the value obtained in the GST-treated extract was divided by the value obtained in the PABP-depleted extract. The stimulation values represent the average ± standard error of three independent duplicate experiments. (B) Krebs-2 cell-free translation reaction mixtures preincubated for 30 min at room temperature with the indicated amounts of purified HIS-Apc5 were programmed with 50 ng of the indicated transcripts for a further 50-min incubation at 37°C. Firefly luciferase activity in the absence of Apc5 was set as 100%. The values represent the average ± standard error of three independent experiments. (C) Krebs-2 cell-free translation reaction mixtures preincubated for 30 min at room tem-perature with 1 μg of HIS-Apc5 and the indicated amounts of purified GST-RRM3 were programmed with 50 ng of the indicated transcripts for a further 50-min incubation at 37°C. Firefly luciferase activity in the absence of HIS-Apc5 and GST-RRM3 was set as 100%. The values represent the average ± standard error of three independent experiments.
Figure Legend Snippet: Effect of PABP and Apc5 on translation in vitro. (A) Krebs-2 cell-free translation reaction mixtures that had been pretreated with GST-Paip2 for PABP depletion or with GST as a control were programmed with 100 ng of the indicated transcripts at 37°C for 90 min followed by measurements of firefly luciferase activity. Immunoblot analysis of the translation extracts using antibodies specific for PABP is shown at the top: lane 1, untreated; lane 2, treated with GST-Paip2; lane 3, treated with GST. For each transcript, the value obtained in the GST-treated extract was divided by the value obtained in the PABP-depleted extract. The stimulation values represent the average ± standard error of three independent duplicate experiments. (B) Krebs-2 cell-free translation reaction mixtures preincubated for 30 min at room temperature with the indicated amounts of purified HIS-Apc5 were programmed with 50 ng of the indicated transcripts for a further 50-min incubation at 37°C. Firefly luciferase activity in the absence of Apc5 was set as 100%. The values represent the average ± standard error of three independent experiments. (C) Krebs-2 cell-free translation reaction mixtures preincubated for 30 min at room tem-perature with 1 μg of HIS-Apc5 and the indicated amounts of purified GST-RRM3 were programmed with 50 ng of the indicated transcripts for a further 50-min incubation at 37°C. Firefly luciferase activity in the absence of HIS-Apc5 and GST-RRM3 was set as 100%. The values represent the average ± standard error of three independent experiments.

Techniques Used: In Vitro, Luciferase, Activity Assay, Purification, Incubation, Transmission Electron Microscopy

11) Product Images from "The Glycosyltransferase Domain of Penicillin-Binding Protein 2a from Streptococcus pneumoniae Catalyzes the Polymerization of Murein Glycan Chains"

Article Title: The Glycosyltransferase Domain of Penicillin-Binding Protein 2a from Streptococcus pneumoniae Catalyzes the Polymerization of Murein Glycan Chains

Journal: Journal of Bacteriology

doi: 10.1128/JB.185.15.4418-4423.2003

Purification of PBP2a*SGG. Proteins were separated by sodium dodecyl sulfate-12.5% PAGE and stained with Coomassie blue. Numbers at the left indicate sizes of standard molecular mass markers. Lanes: 1, molecular mass markers; 2, GST-PBP2a*SGG fusion following purification in a glutathione-Sepharose column; 3, fusion protein cleavage by Tev protease; 4, flowthrough from glutathione-Sepharose (10 μg of protein loaded on the gel); 5, elution from Resource Q column (30 μg of protein loaded on the gel).
Figure Legend Snippet: Purification of PBP2a*SGG. Proteins were separated by sodium dodecyl sulfate-12.5% PAGE and stained with Coomassie blue. Numbers at the left indicate sizes of standard molecular mass markers. Lanes: 1, molecular mass markers; 2, GST-PBP2a*SGG fusion following purification in a glutathione-Sepharose column; 3, fusion protein cleavage by Tev protease; 4, flowthrough from glutathione-Sepharose (10 μg of protein loaded on the gel); 5, elution from Resource Q column (30 μg of protein loaded on the gel).

Techniques Used: Purification, Polyacrylamide Gel Electrophoresis, Staining

Schematic diagrams of S . pneumoniae ). (B) Schematic diagrams of the PBP2a-derived constructs. The peptide at the GST-GT junction of the PBP2a* construct includes sequences specific for thrombin, Tev protease, and factor Xa (italic characters) and N-terminal amino acids of GT (bold characters).
Figure Legend Snippet: Schematic diagrams of S . pneumoniae ). (B) Schematic diagrams of the PBP2a-derived constructs. The peptide at the GST-GT junction of the PBP2a* construct includes sequences specific for thrombin, Tev protease, and factor Xa (italic characters) and N-terminal amino acids of GT (bold characters).

Techniques Used: Derivative Assay, Construct

12) Product Images from "In Vitro and In Vivo Antiangiogenic Properties of the Serpin Protease Nexin-1"

Article Title: In Vitro and In Vivo Antiangiogenic Properties of the Serpin Protease Nexin-1

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.06554-11

Effects of PN-1 on angiogenic responses of HUVECs. (A) Concentration-dependent effect of PN-1 on 10 ng/ml FGF- or VEGF-induced HUVEC proliferation ( n = 4 to 10). (B) Effect of PN-1 (20 μg/ml) on cell proliferation induced by increasing growth factor (GF) concentrations ( n = 4). Results are expressed as the percentage of inhibition of proliferation, calculated from the ratio of the proliferation induced by each growth factor concentration measured in the presence of recombinant PN-1 to that measured in the absence of PN-1. (C and D) HUVEC adhesion (C) and HUVEC spreading on vitronectin and fibronectin (D), in the absence or presence of 20 μg/ml PN-1 ( n = 3 to 4). (E) HUVEC migration in the absence or presence of 20 ng/ml VEGF. *, P
Figure Legend Snippet: Effects of PN-1 on angiogenic responses of HUVECs. (A) Concentration-dependent effect of PN-1 on 10 ng/ml FGF- or VEGF-induced HUVEC proliferation ( n = 4 to 10). (B) Effect of PN-1 (20 μg/ml) on cell proliferation induced by increasing growth factor (GF) concentrations ( n = 4). Results are expressed as the percentage of inhibition of proliferation, calculated from the ratio of the proliferation induced by each growth factor concentration measured in the presence of recombinant PN-1 to that measured in the absence of PN-1. (C and D) HUVEC adhesion (C) and HUVEC spreading on vitronectin and fibronectin (D), in the absence or presence of 20 μg/ml PN-1 ( n = 3 to 4). (E) HUVEC migration in the absence or presence of 20 ng/ml VEGF. *, P

Techniques Used: Concentration Assay, Inhibition, Recombinant, Migration

Effects of PN-1 on HUVEC responses to VEGF. (A) Capillary tube formation in Matrigel in the presence or absence of 50 ng/ml VEGF and 20 μg/ml PN-1. Results are expressed as the mean tube density relative to the control without VEGF, measured on 3 fields per well from 3 to 4 experiments. ***, P
Figure Legend Snippet: Effects of PN-1 on HUVEC responses to VEGF. (A) Capillary tube formation in Matrigel in the presence or absence of 50 ng/ml VEGF and 20 μg/ml PN-1. Results are expressed as the mean tube density relative to the control without VEGF, measured on 3 fields per well from 3 to 4 experiments. ***, P

Techniques Used:

Characterization of PN-1 variants. (Inset) SDS-PAGE Coomassie staining of PN-1 after purification. (A and B) Thrombin inhibition by WT and K7Q PN-1 and catalytic effect of heparin. Thrombin and chromogenic substrate concentrations were 100 pM and 300 μM, respectively. Progress curves are shown. (A) Curves obtained with 50 nM WT (○) and K7Q (□) PN-1, used to determine uncatalyzed rate constants; (B) catalytic effect of 5 nM on thrombin inhibition by 2 nM PN-1. Dashed superimposed lines correspond to WT PN-1, K7Q PN-1, and K7Q PN-1 plus heparin. ●, WT PN1 plus heparin. (C) Representative fluorescence spectra of three of the TNS-bound PN-1 variants in the absence (solid lines) or presence (dashed lines) of heparin, determined with 500 nM WT or K7Q PN-1 and 750 nM R346A. Spectra are difference spectra between TNS plus PN-1 with or without heparin and TNS alone.
Figure Legend Snippet: Characterization of PN-1 variants. (Inset) SDS-PAGE Coomassie staining of PN-1 after purification. (A and B) Thrombin inhibition by WT and K7Q PN-1 and catalytic effect of heparin. Thrombin and chromogenic substrate concentrations were 100 pM and 300 μM, respectively. Progress curves are shown. (A) Curves obtained with 50 nM WT (○) and K7Q (□) PN-1, used to determine uncatalyzed rate constants; (B) catalytic effect of 5 nM on thrombin inhibition by 2 nM PN-1. Dashed superimposed lines correspond to WT PN-1, K7Q PN-1, and K7Q PN-1 plus heparin. ●, WT PN1 plus heparin. (C) Representative fluorescence spectra of three of the TNS-bound PN-1 variants in the absence (solid lines) or presence (dashed lines) of heparin, determined with 500 nM WT or K7Q PN-1 and 750 nM R346A. Spectra are difference spectra between TNS plus PN-1 with or without heparin and TNS alone.

Techniques Used: SDS Page, Staining, Purification, Inhibition, Fluorescence

Impact of PN-1 deficiency on in vivo angiogenesis: vessel formation in Matrigel plugs. Control plugs were implanted in 9 to 10 wild-type mice (PN-1 +/+) or PN-1-deficient mice (PN-1 -/-), and plugs supplemented with 20 μg/ml recombinant PN-1 (rPN-1) were implanted in 3 PN-1-deficient mice (PN-1 −/− + rPN-1). (A) Representative fluorescence of the vessels following retroorbital injection of FITC-dextran. (B) Representative plugs from PN-1 +/+ and PN-1 −/− mice. (C) Representative microphotographs of sections of Matrigel plugs stained with hematoxylin-eosin. Magnification, ×200. Arrows indicate erythrocyte-containing neovessels. (D) Quantification of cell infiltration (4 fields/plug). ***, P
Figure Legend Snippet: Impact of PN-1 deficiency on in vivo angiogenesis: vessel formation in Matrigel plugs. Control plugs were implanted in 9 to 10 wild-type mice (PN-1 +/+) or PN-1-deficient mice (PN-1 -/-), and plugs supplemented with 20 μg/ml recombinant PN-1 (rPN-1) were implanted in 3 PN-1-deficient mice (PN-1 −/− + rPN-1). (A) Representative fluorescence of the vessels following retroorbital injection of FITC-dextran. (B) Representative plugs from PN-1 +/+ and PN-1 −/− mice. (C) Representative microphotographs of sections of Matrigel plugs stained with hematoxylin-eosin. Magnification, ×200. Arrows indicate erythrocyte-containing neovessels. (D) Quantification of cell infiltration (4 fields/plug). ***, P

Techniques Used: In Vivo, Mouse Assay, Recombinant, Fluorescence, Injection, Staining

Impact of PN-1 deficiency on ex vivo angiogenesis. Thoracic aortic rings from PN-1-deficient (-/-) and WT (+/+) mice were cultured in a collagen gel in the presence or absence of 50 ng/ml VEGF and 20 μg/ml recombinant WT PN-1. The microvascular networks sprouting from the rings were observed by phase-contrast microscopy and after isolectin B4 labeling (in red), and results were quantified by densitometric analysis. Representative micrographs and results are shown for 6 to 20 rings from 3 to 7 mice. ***, P
Figure Legend Snippet: Impact of PN-1 deficiency on ex vivo angiogenesis. Thoracic aortic rings from PN-1-deficient (-/-) and WT (+/+) mice were cultured in a collagen gel in the presence or absence of 50 ng/ml VEGF and 20 μg/ml recombinant WT PN-1. The microvascular networks sprouting from the rings were observed by phase-contrast microscopy and after isolectin B4 labeling (in red), and results were quantified by densitometric analysis. Representative micrographs and results are shown for 6 to 20 rings from 3 to 7 mice. ***, P

Techniques Used: Ex Vivo, Mouse Assay, Cell Culture, Recombinant, Microscopy, Labeling

PN-1 binding to HUVECs at 4°C. (A to C) Cells were incubated at 4°C with recombinant PN-1 variants (1 μg/ml) in the presence or not of heparin (Hep), chondroitin sulfate (CS), or dermatan sulfate (DS) (A and C) or with supernatant of resting or activated platelets (B). Whole-cell lysates were submitted to immunoblotting with a polyclonal anti-PN-1 antibody and an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody as a protein loading control. Results are shown with representative immunoblots from at least 3 independent experiments (A, B, and C). (D) Densitometric quantification, expressed as the mean intensity (PN-1/GAPDH ratio) relative to control (WT binding), determined for each immunoblot. NS, not significant; **, P
Figure Legend Snippet: PN-1 binding to HUVECs at 4°C. (A to C) Cells were incubated at 4°C with recombinant PN-1 variants (1 μg/ml) in the presence or not of heparin (Hep), chondroitin sulfate (CS), or dermatan sulfate (DS) (A and C) or with supernatant of resting or activated platelets (B). Whole-cell lysates were submitted to immunoblotting with a polyclonal anti-PN-1 antibody and an anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody as a protein loading control. Results are shown with representative immunoblots from at least 3 independent experiments (A, B, and C). (D) Densitometric quantification, expressed as the mean intensity (PN-1/GAPDH ratio) relative to control (WT binding), determined for each immunoblot. NS, not significant; **, P

Techniques Used: Binding Assay, Incubation, Recombinant, Western Blot

Role of PN-1 functional sites on PN-1 HUVEC angiogenic responses. Effects of PN-1 variants (20 μg/ml [A and C] or 50 μg/ml [B]) on FGF- and VEGF-induced HUVEC proliferation ( n = 4 to 10) (A), VEGF-induced HUVEC migration ( n = 3) (B), and VEGF-induced HUVEC organization in Matrigel (C). The tube density was measured on 3 fields per well from 3 to 4 experiments and averaged relative to the control without PN-1 and VEGF. (D) Effects of WT PN-1 on VEGF 121-induced HUVEC proliferation. **, P
Figure Legend Snippet: Role of PN-1 functional sites on PN-1 HUVEC angiogenic responses. Effects of PN-1 variants (20 μg/ml [A and C] or 50 μg/ml [B]) on FGF- and VEGF-induced HUVEC proliferation ( n = 4 to 10) (A), VEGF-induced HUVEC migration ( n = 3) (B), and VEGF-induced HUVEC organization in Matrigel (C). The tube density was measured on 3 fields per well from 3 to 4 experiments and averaged relative to the control without PN-1 and VEGF. (D) Effects of WT PN-1 on VEGF 121-induced HUVEC proliferation. **, P

Techniques Used: Functional Assay, Migration

13) Product Images from "Activator Protein 2α Associates with Adenomatous Polyposis Coli/β-Catenin and Inhibits β-Catenin/T-cell Factor Transcriptional Activity in Colorectal Cancer Cells *"

Article Title: Activator Protein 2α Associates with Adenomatous Polyposis Coli/β-Catenin and Inhibits β-Catenin/T-cell Factor Transcriptional Activity in Colorectal Cancer Cells *

Journal: The Journal of biological chemistry

doi: 10.1074/jbc.M405025200

Truncated forms of AP-2 α  fail to inhibit TOPflash reporter activity A , schematic presentation of full-length and truncated forms of AP-2 α  transfected into HEK293 cells with TOPflash,  β -galactosidase, and S33Y  β -catenin ( B ) or no exogenous  β -catenin ( C ). Lysates were prepared and analyzed for luciferase and  β -galactosidase activities 48 h post-transfection. Luciferase activity was normalized to the corresponding  β -galactosidase activity to obtain the relative luciferase activity. Data are presented as mean ± S.D.,  n  = 3.
Figure Legend Snippet: Truncated forms of AP-2 α fail to inhibit TOPflash reporter activity A , schematic presentation of full-length and truncated forms of AP-2 α transfected into HEK293 cells with TOPflash, β -galactosidase, and S33Y β -catenin ( B ) or no exogenous β -catenin ( C ). Lysates were prepared and analyzed for luciferase and β -galactosidase activities 48 h post-transfection. Luciferase activity was normalized to the corresponding β -galactosidase activity to obtain the relative luciferase activity. Data are presented as mean ± S.D., n = 3.

Techniques Used: Activity Assay, Transfection, Luciferase

AP-2 α  binds to the N-terminal repeats in APC A , scheme showing wild type (full-length) APC and truncated proteins (APC1-APC5) with one or two of the repeat domains. *, positions of nuclear export signals.  B ,  35 S-labeled APC fragments as well as  β -catenin were synthesized  in vitro  and incubated with Sepharose-bound GST-AP-2 α . Beads were washed, and bound  35 S-labeled proteins were eluted, followed by SDS-PAGE and autoradiography. The  lower panel  shows 50% of the total radioactivity in each reaction (50% input controls).
Figure Legend Snippet: AP-2 α binds to the N-terminal repeats in APC A , scheme showing wild type (full-length) APC and truncated proteins (APC1-APC5) with one or two of the repeat domains. *, positions of nuclear export signals. B , 35 S-labeled APC fragments as well as β -catenin were synthesized in vitro and incubated with Sepharose-bound GST-AP-2 α . Beads were washed, and bound 35 S-labeled proteins were eluted, followed by SDS-PAGE and autoradiography. The lower panel shows 50% of the total radioactivity in each reaction (50% input controls).

Techniques Used: Labeling, Synthesized, In Vitro, Incubation, SDS Page, Autoradiography, Radioactivity

TOPflash reporter activation by exogenous β -catenin is suppressed by AP-2 α in HEK293 cells Cells were transfected with TOPflash, a β -catenin/TCF/LEF-responsive luciferase reporter plasmid containing TCF-4-binding sites, or the corresponding negative control FOPflash, as well as with AP-2 α and β -catenin constructs (as indicated in the figure). pSV- β -Gal was used as internal control. Luciferase and β -galactosidase activities were determined for whole cell lysates 48 h post-transfection, as described under “Experimental Procedures.” Luciferase activity was normalized to the corresponding β -galactosidase activity to obtain the relative luciferase activity. Data are presented as mean ± S.D., n = 3. A , induction of TOPflash activity by wild type β -catenin is suppressed by AP-2 α . The cell lysates also were immunoblotted for AP-2 α , β -catenin, and β -actin ( lower panel ). B , overexpression of AP-2 α inhibits TOPflash activity induced by oncogenic S33Y β -catenin. C , AP-2 α inhibits induction of TOPflash activity by Δ45 β -catenin.
Figure Legend Snippet: TOPflash reporter activation by exogenous β -catenin is suppressed by AP-2 α in HEK293 cells Cells were transfected with TOPflash, a β -catenin/TCF/LEF-responsive luciferase reporter plasmid containing TCF-4-binding sites, or the corresponding negative control FOPflash, as well as with AP-2 α and β -catenin constructs (as indicated in the figure). pSV- β -Gal was used as internal control. Luciferase and β -galactosidase activities were determined for whole cell lysates 48 h post-transfection, as described under “Experimental Procedures.” Luciferase activity was normalized to the corresponding β -galactosidase activity to obtain the relative luciferase activity. Data are presented as mean ± S.D., n = 3. A , induction of TOPflash activity by wild type β -catenin is suppressed by AP-2 α . The cell lysates also were immunoblotted for AP-2 α , β -catenin, and β -actin ( lower panel ). B , overexpression of AP-2 α inhibits TOPflash activity induced by oncogenic S33Y β -catenin. C , AP-2 α inhibits induction of TOPflash activity by Δ45 β -catenin.

Techniques Used: Activation Assay, Transfection, Luciferase, Plasmid Preparation, Binding Assay, Negative Control, Construct, Activity Assay, Over Expression

Nuclear β -catenin and TCF-4 levels are unchanged by AP-2 α Cells were transfected with plasmid pcDNA3.1/AP-2 α or empty vector pcDNA3.1(+). After 48 h, nuclear lysates were prepared and immunoblotted for β -catenin, TCF-4, and AP-2 α , using β -actin as loading control.
Figure Legend Snippet: Nuclear β -catenin and TCF-4 levels are unchanged by AP-2 α Cells were transfected with plasmid pcDNA3.1/AP-2 α or empty vector pcDNA3.1(+). After 48 h, nuclear lysates were prepared and immunoblotted for β -catenin, TCF-4, and AP-2 α , using β -actin as loading control.

Techniques Used: Transfection, Plasmid Preparation

AP-2 α associates with APC and β -catenin, but not with TCF-4 Cell lysates from nontransfected cells were normalized for protein content and subjected to immunoprecipitation ( IP ) followed by immunoblotting ( IB ). A , immunoprecipitation was performed with the antibodies indicated, followed by immunoblotting with anti-TCF-4 antibody. Negative controls included no antibody ( No Ab ) and irrelevant antibody ( HA-Tag ). Note that AP-2 α antibody failed to pull down TCF-4. B , immunoprecipitation was first performed with the antibodies indicated, followed by immunoblotting with anti- β -catenin. β -Catenin was associated both with APC and with AP-2 α. C , immunoprecipitation with the antibodies indicated followed by immunoblotting with anti-APC identified full-length APC bound to AP-2 α in HEK293 and HCT116 cells. D , in HT29 cells, immunoprecipitation with anti-AP-2 α antibody pulled down 200- and 100-kDa truncated forms of APC, and anti-APC antibody pulled down AP-2 α (48 kDa).
Figure Legend Snippet: AP-2 α associates with APC and β -catenin, but not with TCF-4 Cell lysates from nontransfected cells were normalized for protein content and subjected to immunoprecipitation ( IP ) followed by immunoblotting ( IB ). A , immunoprecipitation was performed with the antibodies indicated, followed by immunoblotting with anti-TCF-4 antibody. Negative controls included no antibody ( No Ab ) and irrelevant antibody ( HA-Tag ). Note that AP-2 α antibody failed to pull down TCF-4. B , immunoprecipitation was first performed with the antibodies indicated, followed by immunoblotting with anti- β -catenin. β -Catenin was associated both with APC and with AP-2 α. C , immunoprecipitation with the antibodies indicated followed by immunoblotting with anti-APC identified full-length APC bound to AP-2 α in HEK293 and HCT116 cells. D , in HT29 cells, immunoprecipitation with anti-AP-2 α antibody pulled down 200- and 100-kDa truncated forms of APC, and anti-APC antibody pulled down AP-2 α (48 kDa).

Techniques Used: Immunoprecipitation

AP-2 α  and APC, alone or in combination, inhibits the interaction of  β -catenin with TCF-4 in the nucleus AP-2 α  and/or APC were overexpressed by transient transfection, as indicated, and anti-TCF-4 antibody was used to pull-down  β -catenin from nuclear extracts, 48 h post-transfection.  β -Catenin levels were analyzed by immunoblotting, and the same blots were reprobed for TCF-4. Nuclear lysates from control groups were used for negative controls of co-immu-noprecipitation, including no antibody ( No Ab ) and irrelevant antibody ( HA-Tag ). Relative  β -catenin levels were determined by densitometric analysis and normalized to TCF-4;  β -catenin expression recovered in the nontransfected controls was assigned an arbitrary value of 1.00.
Figure Legend Snippet: AP-2 α and APC, alone or in combination, inhibits the interaction of β -catenin with TCF-4 in the nucleus AP-2 α and/or APC were overexpressed by transient transfection, as indicated, and anti-TCF-4 antibody was used to pull-down β -catenin from nuclear extracts, 48 h post-transfection. β -Catenin levels were analyzed by immunoblotting, and the same blots were reprobed for TCF-4. Nuclear lysates from control groups were used for negative controls of co-immu-noprecipitation, including no antibody ( No Ab ) and irrelevant antibody ( HA-Tag ). Relative β -catenin levels were determined by densitometric analysis and normalized to TCF-4; β -catenin expression recovered in the nontransfected controls was assigned an arbitrary value of 1.00.

Techniques Used: Transfection, Expressing

14) Product Images from "SGEF, a RhoG Guanine Nucleotide Exchange Factor that Stimulates Macropinocytosis"

Article Title: SGEF, a RhoG Guanine Nucleotide Exchange Factor that Stimulates Macropinocytosis

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E04-02-0146

Myc-SGEF stimulates RhoG activation in NIH3T3 cells. GST-PBD was used to pull down GTP-loaded Rac1 or Cdc42. GST-ELMO was used to pull down GTP-loaded Myc-RhoG. Low-level expression of Myc-RhoG was used in place of the endogenous protein for lack of a specific RhoG antibody. Myc-SGEF, Myc-SGEFΔN, or GFP-onco-Vav2 (positive control) was transiently expressed and the resulting change in GTPase activation (labeled active) compared with empty vector-transfected fibroblasts (Mock).
Figure Legend Snippet: Myc-SGEF stimulates RhoG activation in NIH3T3 cells. GST-PBD was used to pull down GTP-loaded Rac1 or Cdc42. GST-ELMO was used to pull down GTP-loaded Myc-RhoG. Low-level expression of Myc-RhoG was used in place of the endogenous protein for lack of a specific RhoG antibody. Myc-SGEF, Myc-SGEFΔN, or GFP-onco-Vav2 (positive control) was transiently expressed and the resulting change in GTPase activation (labeled active) compared with empty vector-transfected fibroblasts (Mock).

Techniques Used: Activation Assay, Expressing, Positive Control, Labeling, Plasmid Preparation, Transfection

15) Product Images from "The adaptor protein p40phox as a positive regulator of the superoxide-producing phagocyte oxidase"

Article Title: The adaptor protein p40phox as a positive regulator of the superoxide-producing phagocyte oxidase

Journal: The EMBO Journal

doi: 10.1093/emboj/cdf642

Fig. 8 . p40 phox -enhanced superoxide production upon cell stimulation with the muscarinic receptor peptide m4I3C(14). ( A ) Superoxide production by the K562 cells with stable expression of p40 phox or without p40 phox . The K562 cells (5.0 × 10 3 cells) were stimulated with the muscarinic receptor peptide m4I3C(14) (m) (200 µM) and the chemiluminescence change was continuously monitored with DIOGENES, and SOD (50 µg/ml) was added where indicated (arrowhead). For details see Materials and methods. ( B ) Effect of pertussis toxin (PTX) on superoxide production upon stimulation with m4I3C(14) or PMA. The K562 cells (5.0 × 10 3 cells) were pretreated with PTX (18 µg/ml) and stimulated with m4I3C(14) (200 µM) or PMA (200 ng/ml) and superoxide production was measured as change of chemiluminescence. Each histogram indicates the average from five independent experiments, with bars representing SD. ( C ) Effect of the D289A substitution in the PC motif of p40 phox on superoxide production upon stimulation with m4I3C(14). The K562 cells with p40 phox (WT) or p40 phox (D289A) or without p40 phox (5.0 × 10 3 cells) were stimulated with 200 µM m4I3C(14) and superoxide production was measured as change of chemiluminescence. Each histogram represents the average from five independent experiments, with bars representing SD. ( D ) Effect of the K355A substitution in the PB1 domain of p67 phox on superoxide production upon stimulation with m4I3C(14). The K562 cells with p67 phox (WT) or p67 phox (K355A) or without p67 phox (5.0 × 10 3 cells) were stimulated with 200 µM m4I3C(14), and superoxide production was measured as change of chemiluminescence. Each histogram indicates the average from five independent experiments, with bars representing SD.
Figure Legend Snippet: Fig. 8 . p40 phox -enhanced superoxide production upon cell stimulation with the muscarinic receptor peptide m4I3C(14). ( A ) Superoxide production by the K562 cells with stable expression of p40 phox or without p40 phox . The K562 cells (5.0 × 10 3 cells) were stimulated with the muscarinic receptor peptide m4I3C(14) (m) (200 µM) and the chemiluminescence change was continuously monitored with DIOGENES, and SOD (50 µg/ml) was added where indicated (arrowhead). For details see Materials and methods. ( B ) Effect of pertussis toxin (PTX) on superoxide production upon stimulation with m4I3C(14) or PMA. The K562 cells (5.0 × 10 3 cells) were pretreated with PTX (18 µg/ml) and stimulated with m4I3C(14) (200 µM) or PMA (200 ng/ml) and superoxide production was measured as change of chemiluminescence. Each histogram indicates the average from five independent experiments, with bars representing SD. ( C ) Effect of the D289A substitution in the PC motif of p40 phox on superoxide production upon stimulation with m4I3C(14). The K562 cells with p40 phox (WT) or p40 phox (D289A) or without p40 phox (5.0 × 10 3 cells) were stimulated with 200 µM m4I3C(14) and superoxide production was measured as change of chemiluminescence. Each histogram represents the average from five independent experiments, with bars representing SD. ( D ) Effect of the K355A substitution in the PB1 domain of p67 phox on superoxide production upon stimulation with m4I3C(14). The K562 cells with p67 phox (WT) or p67 phox (K355A) or without p67 phox (5.0 × 10 3 cells) were stimulated with 200 µM m4I3C(14), and superoxide production was measured as change of chemiluminescence. Each histogram indicates the average from five independent experiments, with bars representing SD.

Techniques Used: Cell Stimulation, Expressing

Fig. 6 . Effect of the K355A substitution in the PB1 domain of p67 phox on its interaction with p40 phox . ( A ) Expression of p67 phox in K562 cells. K562 cells stably expressing gp91 phox , p47 phox and p40 phox were transfected with pREP4 encoding p67 phox (WT) or p67 phox (K355A). The K562 cells were stained with anti-p67 phox serum (filled histogram) or pre-immune serum (open histogram), and analyzed by flow cytometry. ( B ) In vivo interaction of p67 phox with p40 phox in the K562 cells. The cell lysates of the K562 cells were analyzed by immunoprecipitation with the anti-p67 phox or control IgG (cont.) (left panel), or the anti-p47 phox antibody or control IgG (cont.) (right panel) followed by immunoblot (Blot) with the indicated antibody.
Figure Legend Snippet: Fig. 6 . Effect of the K355A substitution in the PB1 domain of p67 phox on its interaction with p40 phox . ( A ) Expression of p67 phox in K562 cells. K562 cells stably expressing gp91 phox , p47 phox and p40 phox were transfected with pREP4 encoding p67 phox (WT) or p67 phox (K355A). The K562 cells were stained with anti-p67 phox serum (filled histogram) or pre-immune serum (open histogram), and analyzed by flow cytometry. ( B ) In vivo interaction of p67 phox with p40 phox in the K562 cells. The cell lysates of the K562 cells were analyzed by immunoprecipitation with the anti-p67 phox or control IgG (cont.) (left panel), or the anti-p47 phox antibody or control IgG (cont.) (right panel) followed by immunoblot (Blot) with the indicated antibody.

Techniques Used: Expressing, Stable Transfection, Transfection, Staining, Flow Cytometry, Cytometry, In Vivo, Immunoprecipitation

Fig. 7 . Effect of the K355A substitution in the PB1 domain on membrane translocation of p47 phox and p67 phox and superoxide production upon PMA stimulation. ( A ) Membrane translocation of p47 phox , p67 phox and p40 phox upon PMA stimulation. The K562 cells stably expressing p67 phox (WT) or p67 phox (K355A) (1.0 × 10 7 cells) were stimulated with PMA (200 ng/ml) for the indicated time and the amounts of p47 phox , p67 phox and p40 phox in the membrane fractions were analyzed by immunoblot. ( B ) Superoxide production by the K562 cells with p67 phox (WT) or p67 phox (K355A) was measured as change of chemiluminescence. The K562 cells (5.0 × 10 3 cells) were stimulated with PMA (200 ng/ml) and the chemiluminescence change was measured. The chemiluminescence change by K562 cells without the expression of p40 phox and with p67 phox (WT) or p67 phox (K355A) was also measured. Each histogram indicates the average from five independent experiments, with bars representing SD.
Figure Legend Snippet: Fig. 7 . Effect of the K355A substitution in the PB1 domain on membrane translocation of p47 phox and p67 phox and superoxide production upon PMA stimulation. ( A ) Membrane translocation of p47 phox , p67 phox and p40 phox upon PMA stimulation. The K562 cells stably expressing p67 phox (WT) or p67 phox (K355A) (1.0 × 10 7 cells) were stimulated with PMA (200 ng/ml) for the indicated time and the amounts of p47 phox , p67 phox and p40 phox in the membrane fractions were analyzed by immunoblot. ( B ) Superoxide production by the K562 cells with p67 phox (WT) or p67 phox (K355A) was measured as change of chemiluminescence. The K562 cells (5.0 × 10 3 cells) were stimulated with PMA (200 ng/ml) and the chemiluminescence change was measured. The chemiluminescence change by K562 cells without the expression of p40 phox and with p67 phox (WT) or p67 phox (K355A) was also measured. Each histogram indicates the average from five independent experiments, with bars representing SD.

Techniques Used: Translocation Assay, Stable Transfection, Expressing

16) Product Images from "Regulated protein degradation controls PKA function and cell-type differentiation in Dictyostelium"

Article Title: Regulated protein degradation controls PKA function and cell-type differentiation in Dictyostelium

Journal: Genes & Development

doi: 10.1101/gad.871101

Western blots of RegA, PKAcat, and PKA-R in various cell lines. ( A ). ( B ) Wild-type and mutant strains expressing GST–FbxA:F-box/WD40 were lysed and the 10,000 × G supernatant was adsorbed to g–Sepharose. The beads were washed and the bound material was examined by Western blot analysis and probed with anti-RegA, Cul-1, and GST antibodies (see Materials and Methods). ( C ) Samples were taken and processed as described for A and probed with either anti-PKAcat, or anti-PKA-R antibodies, as indicated. The anti-PKAcat and anti-PKA-R antibodies were a generous gift of M. Veron (Institut Pasteur, Paris). ( D ) The experiment is the same as described in B except that it was performed using cells that were transformed with GST alone.
Figure Legend Snippet: Western blots of RegA, PKAcat, and PKA-R in various cell lines. ( A ). ( B ) Wild-type and mutant strains expressing GST–FbxA:F-box/WD40 were lysed and the 10,000 × G supernatant was adsorbed to g–Sepharose. The beads were washed and the bound material was examined by Western blot analysis and probed with anti-RegA, Cul-1, and GST antibodies (see Materials and Methods). ( C ) Samples were taken and processed as described for A and probed with either anti-PKAcat, or anti-PKA-R antibodies, as indicated. The anti-PKAcat and anti-PKA-R antibodies were a generous gift of M. Veron (Institut Pasteur, Paris). ( D ) The experiment is the same as described in B except that it was performed using cells that were transformed with GST alone.

Techniques Used: Western Blot, Mutagenesis, Expressing, Transformation Assay

17) Product Images from "The Yeast Eukaryotic Initiation Factor 4G (eIF4G) HEAT Domain Interacts with eIF1 and eIF5 and Is Involved in Stringent AUG Selection"

Article Title: The Yeast Eukaryotic Initiation Factor 4G (eIF4G) HEAT Domain Interacts with eIF1 and eIF5 and Is Involved in Stringent AUG Selection

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.15.5431-5445.2003

Purified eIF4A does not inhibit interaction between eIF4G2 HEAT domain and eIF1 or eIF5-CTD in vitro. 35 S-eIF4G2 439-914 was bound to GST fusion proteins (10 μg), indicated across the top, in 200 μl of binding buffer in the presence or absence of ATP (1 mM) or eIF4A (80 μg) and analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie staining (top panel) or autoradiography (bottom panel). Lane 1 indicates the input amount of 35 S-eIF4G2 439-914 used in this study. The percentage of 35 S-eIF4G2 439-914 bound to each GST fusion protein is shown below the autoradiography.
Figure Legend Snippet: Purified eIF4A does not inhibit interaction between eIF4G2 HEAT domain and eIF1 or eIF5-CTD in vitro. 35 S-eIF4G2 439-914 was bound to GST fusion proteins (10 μg), indicated across the top, in 200 μl of binding buffer in the presence or absence of ATP (1 mM) or eIF4A (80 μg) and analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie staining (top panel) or autoradiography (bottom panel). Lane 1 indicates the input amount of 35 S-eIF4G2 439-914 used in this study. The percentage of 35 S-eIF4G2 439-914 bound to each GST fusion protein is shown below the autoradiography.

Techniques Used: Purification, In Vitro, Binding Assay, Polyacrylamide Gel Electrophoresis, Staining, Autoradiography

Effect of deletions (A) and point mutations (B) on eIF4G2 binding to eIF1 and eIF5. The binding of GST (column C, lane 2), GST-eIF1 (column 1, lane 3), or GST-eIF5 241-405 (column 5, lane 4) (20 μg each) to different derivatives of 35 S-eIF4G2 synthesized in a rabbit reticulocyte lysate was assayed as described in Materials and Methods. The empty box at the top denotes the primary structure of S. cerevisiae ) and the putative primary binding site for eIF1 and eIF5 deduced from this study. The thick lines below describe the segments of eIF4G2 with their clone designations and locations in the sequence. Each panel on the right shows the autoradiography of the 35 S-labeled derivative of eIF4G2 drawn in the diagram, with lane 1 (In) showing a 20% input amount, and the table in the middle summarizes the percentage of 35 S-labeled proteins bound to GST fusion proteins. Empty circles indicate the positions of amino acid substitutions in each tif4632 allele used.
Figure Legend Snippet: Effect of deletions (A) and point mutations (B) on eIF4G2 binding to eIF1 and eIF5. The binding of GST (column C, lane 2), GST-eIF1 (column 1, lane 3), or GST-eIF5 241-405 (column 5, lane 4) (20 μg each) to different derivatives of 35 S-eIF4G2 synthesized in a rabbit reticulocyte lysate was assayed as described in Materials and Methods. The empty box at the top denotes the primary structure of S. cerevisiae ) and the putative primary binding site for eIF1 and eIF5 deduced from this study. The thick lines below describe the segments of eIF4G2 with their clone designations and locations in the sequence. Each panel on the right shows the autoradiography of the 35 S-labeled derivative of eIF4G2 drawn in the diagram, with lane 1 (In) showing a 20% input amount, and the table in the middle summarizes the percentage of 35 S-labeled proteins bound to GST fusion proteins. Empty circles indicate the positions of amino acid substitutions in each tif4632 allele used.

Techniques Used: Binding Assay, Synthesized, Sequencing, Autoradiography, Labeling

Yeast eIF4G interacts with eIF1 and eIF5 in vitro. (A) Microtiter plate binding assay with 1 μg of purified eIF4G1 incubated with 0.5 μg of immobilized BSA (column 1) or eIF1 (column 2). (B) Microtiter plate assay with 10 pmol of purified GST, GST-eIF1, or GST-eIF5 incubated with 10 pmol of immobilized BSA (columns 1, 4, and 7), eIF4G2 1-513 (columns 2, 5, and 8), or eIF4G2 439-914 (columns 3, 6, and 9). The relative amount of bound eIF4G1 or GST-fused proteins was indirectly determined by A 405 , shown on the y axis, resulting from the reaction catalyzed by alkaline phosphatase-conjugated secondary antibodies that were added to the reaction together with primary antibodies, indicated to the left. Error bars refer to the standard deviation from six wells. (C) GST pull-down assays with purified eIF4G2 439-914 . A 200-pmol aliquot of eIF4G2 439-914 was incubated with equimolar GST (lane 1), GST-eIF1 (lanes 2 and 3), or GST-eIF5 (lanes 4 and 5) in 160 μl of HBS containing glutathione-agarose resin (Sigma) and 1 μg of RNase A/ml (lanes 3 and 5), where indicated, for 30 min at 4°C. After washing the resin three times with 1 ml of HBS, bound proteins were eluted with 70 μl of a 30 mM glutathione solution in 0.2 M Tris-HCl (pH 7.5). Portions of eluates (bottom panel) and protein mixtures prior to binding to the glutathione resin (top panel) were analyzed with SDS-polyacrylamide gel electrophoresis and silver staining. The asterisk indicates a degradation product of GST-eIF5. (D) GST pull-down assays with 35 S-eIF4G2 439-914 . The binding of different amounts of GST-eIF1 and GST-eIF5 241-405 to 35 S-eIF4G2 439-914 ). Autoradiography of bound 35 S-eIF4G2 439-914 is shown on top (lanes 3 to 12). Lane 1, 20% input amount; lane 2, bound fraction with no GST fusion protein on the resin. The fraction of bound 35 S-eIF4G2 439-914 was plotted against the molar concentration of GST fusion proteins. Numbers in the graph refer to lane numbers for the autoradiography. The K d values given in the text are the averages of those calculated from each point.
Figure Legend Snippet: Yeast eIF4G interacts with eIF1 and eIF5 in vitro. (A) Microtiter plate binding assay with 1 μg of purified eIF4G1 incubated with 0.5 μg of immobilized BSA (column 1) or eIF1 (column 2). (B) Microtiter plate assay with 10 pmol of purified GST, GST-eIF1, or GST-eIF5 incubated with 10 pmol of immobilized BSA (columns 1, 4, and 7), eIF4G2 1-513 (columns 2, 5, and 8), or eIF4G2 439-914 (columns 3, 6, and 9). The relative amount of bound eIF4G1 or GST-fused proteins was indirectly determined by A 405 , shown on the y axis, resulting from the reaction catalyzed by alkaline phosphatase-conjugated secondary antibodies that were added to the reaction together with primary antibodies, indicated to the left. Error bars refer to the standard deviation from six wells. (C) GST pull-down assays with purified eIF4G2 439-914 . A 200-pmol aliquot of eIF4G2 439-914 was incubated with equimolar GST (lane 1), GST-eIF1 (lanes 2 and 3), or GST-eIF5 (lanes 4 and 5) in 160 μl of HBS containing glutathione-agarose resin (Sigma) and 1 μg of RNase A/ml (lanes 3 and 5), where indicated, for 30 min at 4°C. After washing the resin three times with 1 ml of HBS, bound proteins were eluted with 70 μl of a 30 mM glutathione solution in 0.2 M Tris-HCl (pH 7.5). Portions of eluates (bottom panel) and protein mixtures prior to binding to the glutathione resin (top panel) were analyzed with SDS-polyacrylamide gel electrophoresis and silver staining. The asterisk indicates a degradation product of GST-eIF5. (D) GST pull-down assays with 35 S-eIF4G2 439-914 . The binding of different amounts of GST-eIF1 and GST-eIF5 241-405 to 35 S-eIF4G2 439-914 ). Autoradiography of bound 35 S-eIF4G2 439-914 is shown on top (lanes 3 to 12). Lane 1, 20% input amount; lane 2, bound fraction with no GST fusion protein on the resin. The fraction of bound 35 S-eIF4G2 439-914 was plotted against the molar concentration of GST fusion proteins. Numbers in the graph refer to lane numbers for the autoradiography. The K d values given in the text are the averages of those calculated from each point.

Techniques Used: In Vitro, Binding Assay, Purification, Incubation, Standard Deviation, Polyacrylamide Gel Electrophoresis, Silver Staining, Autoradiography, Concentration Assay

). Thick arrows indicate the subunits of eIFs that bind to Met-tRNAiMet, mRNA, or the 40S ribosomal subunit. (B and C) GST fusion proteins indicated across the top were bound to 35 S-eIF1 (B) or 35 S-eIF5 241-405 ) containing 20 μg of eIF4G2 439-914 or eIF3c 1-156 as indicated. GST fusion-containing complexes (lanes 3 to 10), together with 10 and 20% input amounts of the reaction mixtures (lanes 1 and 2, respectively), were analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie staining (top panels) and autoradiography (bottom panels). (D and E) GST fusion proteins indicated across the top were bound to 35 S-eIF5 241-405 in the presence of bacterial cell extracts containing specified amounts of eIF1 (D) or bound to 35 S-eIF4G2 439-914 in the presence of bacterial cell extracts induced for recombinant eIF1 (12 μg) from pT7-SUI1 (I) or those mock induced from cells containing an empty vector pT7-7 (U) (E). All the binding reactions were conducted with the indicated 35 S-labeled proteins similarly to those shown in panels B and C. Lanes 1, 20% of input amounts of 35 S-labeled proteins. Brackets indicate milk proteins in the binding buffer that occasionally attach to the glutathione resin. The appearance of these proteins did not have any effect on the results of the experiments. The percentages of 35 S-labeled proteins bound to GST fusion proteins are indicated below the bottom portions of panels B to E.
Figure Legend Snippet: ). Thick arrows indicate the subunits of eIFs that bind to Met-tRNAiMet, mRNA, or the 40S ribosomal subunit. (B and C) GST fusion proteins indicated across the top were bound to 35 S-eIF1 (B) or 35 S-eIF5 241-405 ) containing 20 μg of eIF4G2 439-914 or eIF3c 1-156 as indicated. GST fusion-containing complexes (lanes 3 to 10), together with 10 and 20% input amounts of the reaction mixtures (lanes 1 and 2, respectively), were analyzed by SDS-polyacrylamide gel electrophoresis followed by Coomassie staining (top panels) and autoradiography (bottom panels). (D and E) GST fusion proteins indicated across the top were bound to 35 S-eIF5 241-405 in the presence of bacterial cell extracts containing specified amounts of eIF1 (D) or bound to 35 S-eIF4G2 439-914 in the presence of bacterial cell extracts induced for recombinant eIF1 (12 μg) from pT7-SUI1 (I) or those mock induced from cells containing an empty vector pT7-7 (U) (E). All the binding reactions were conducted with the indicated 35 S-labeled proteins similarly to those shown in panels B and C. Lanes 1, 20% of input amounts of 35 S-labeled proteins. Brackets indicate milk proteins in the binding buffer that occasionally attach to the glutathione resin. The appearance of these proteins did not have any effect on the results of the experiments. The percentages of 35 S-labeled proteins bound to GST fusion proteins are indicated below the bottom portions of panels B to E.

Techniques Used: Polyacrylamide Gel Electrophoresis, Staining, Autoradiography, Recombinant, Plasmid Preparation, Binding Assay, Labeling

18) Product Images from "IEX-1: a new ERK substrate involved in both ERK survival activity and ERK activation"

Article Title: IEX-1: a new ERK substrate involved in both ERK survival activity and ERK activation

Journal: The EMBO Journal

doi: 10.1093/emboj/cdf488

Fig. 3. IEX-1 binds specifically to the active forms of ERK1/2. ( A and B ) Sepharose-bound GST or GST–IEX-1 wild type and mutants were incubated with lysates from UT7 cells either untreated (0) or stimulated with 10 nM TPO or 100 ng/ml anisomycin (Aniso) for 30 min, as indicated. MAPKs were detected in GST precipitates or in samples of total lysates (TL) by immunoblotting with antibodies directed against the active forms of ERK, JNK or p38. ( C and D ) Cos7 cells were transfected with 2 µg of pcDNA-HA-IEX-1 (Wt or ΔBD mutant) or empty vector (V), starved overnight and stimulated (+) or not (–) with 100 ng/ml EGF for 10 min. Lysates were immunoprecipitated (IP) with anti-HA or anti-ERK1 antibodies and analyzed by western blotting. Expressions of ERK and HA-IEX-1 are shown in total lysates (TL). ( E ) UT7 cells (50 × 10 7 ) were treated with TPO for 3 h to induce endogenous IEX-1 protein expression. The presence of IEX-1 and phosphorylated ERK was monitored in anti-IEX-1, anti-phosphoERK or control (C) immunoprecipitates (IP), as indicated. As a control, phosphoERK and IEX-1 expressions are shown in total lysates (TL) from 1 × 10 6 cells.
Figure Legend Snippet: Fig. 3. IEX-1 binds specifically to the active forms of ERK1/2. ( A and B ) Sepharose-bound GST or GST–IEX-1 wild type and mutants were incubated with lysates from UT7 cells either untreated (0) or stimulated with 10 nM TPO or 100 ng/ml anisomycin (Aniso) for 30 min, as indicated. MAPKs were detected in GST precipitates or in samples of total lysates (TL) by immunoblotting with antibodies directed against the active forms of ERK, JNK or p38. ( C and D ) Cos7 cells were transfected with 2 µg of pcDNA-HA-IEX-1 (Wt or ΔBD mutant) or empty vector (V), starved overnight and stimulated (+) or not (–) with 100 ng/ml EGF for 10 min. Lysates were immunoprecipitated (IP) with anti-HA or anti-ERK1 antibodies and analyzed by western blotting. Expressions of ERK and HA-IEX-1 are shown in total lysates (TL). ( E ) UT7 cells (50 × 10 7 ) were treated with TPO for 3 h to induce endogenous IEX-1 protein expression. The presence of IEX-1 and phosphorylated ERK was monitored in anti-IEX-1, anti-phosphoERK or control (C) immunoprecipitates (IP), as indicated. As a control, phosphoERK and IEX-1 expressions are shown in total lysates (TL) from 1 × 10 6 cells.

Techniques Used: Incubation, Transfection, Mutagenesis, Plasmid Preparation, Immunoprecipitation, Western Blot, Expressing

Fig. 7. The ability of IEX-1 to stimulate ERK activity is specific and requires its DEF motif. ( A ) CHO cells were transfected with HA-IEX-1 together with HA-JNK1 or empty vector (V) and treated or not for 10 min with 100 ng/ml anisomycin. Activated JNK was detected in anti-HA immunoprecipitates by immunoblotting with anti-phospho-JNK antibodies and by in vitro kinase assay using GST–c-jun as substrate. ( B and C ) CHO cells were transfected with HA-ERK, along with either empty vector (V), plasmids encoding the indicated HA-IEX-1 species or HA-Elk1. ERK activity was determined in anti-HA immunoprecipitates by in vitro kinase assay.
Figure Legend Snippet: Fig. 7. The ability of IEX-1 to stimulate ERK activity is specific and requires its DEF motif. ( A ) CHO cells were transfected with HA-IEX-1 together with HA-JNK1 or empty vector (V) and treated or not for 10 min with 100 ng/ml anisomycin. Activated JNK was detected in anti-HA immunoprecipitates by immunoblotting with anti-phospho-JNK antibodies and by in vitro kinase assay using GST–c-jun as substrate. ( B and C ) CHO cells were transfected with HA-ERK, along with either empty vector (V), plasmids encoding the indicated HA-IEX-1 species or HA-Elk1. ERK activity was determined in anti-HA immunoprecipitates by in vitro kinase assay.

Techniques Used: Activity Assay, Transfection, Plasmid Preparation, In Vitro, Kinase Assay

Fig. 1. ERKs phosphorylate IEX-1 in vitro . ( A ) One microgram of purified GST or GST–IEX-1 fusion proteins wild-type (Wt) and mutants (T18A; T123AS126A; T/SA3; ΔBD) were reacted with purified recombinant active ERK2 in the presence of [ 32 P]ATP. The products were analyzed by autoradiography or western blotting (WB) with the indicated antibodies. ( B ) Schematic representation of IEX-1 Wt and ERK-phosphorylation and/or binding sites mutants. Hatched area indicates the putative IEX-1 transmembrane domain.
Figure Legend Snippet: Fig. 1. ERKs phosphorylate IEX-1 in vitro . ( A ) One microgram of purified GST or GST–IEX-1 fusion proteins wild-type (Wt) and mutants (T18A; T123AS126A; T/SA3; ΔBD) were reacted with purified recombinant active ERK2 in the presence of [ 32 P]ATP. The products were analyzed by autoradiography or western blotting (WB) with the indicated antibodies. ( B ) Schematic representation of IEX-1 Wt and ERK-phosphorylation and/or binding sites mutants. Hatched area indicates the putative IEX-1 transmembrane domain.

Techniques Used: In Vitro, Purification, Recombinant, Autoradiography, Western Blot, Binding Assay

19) Product Images from "Modulation of Retinoid Signaling by a Cytoplasmic Viral Protein via Sequestration of Sp110b, a Potent Transcriptional Corepressor of Retinoic Acid Receptor, from the Nucleus"

Article Title: Modulation of Retinoid Signaling by a Cytoplasmic Viral Protein via Sequestration of Sp110b, a Potent Transcriptional Corepressor of Retinoic Acid Receptor, from the Nucleus

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.23.21.7498-7509.2003

Interaction of the core with Sp110b. (A) The 35 S-labeled in vitro transcription-translation product of full-length core (top panel) or the core(6162M) mutant (center panel) was incubated with recombinant Sp110 or Sp110b fused to GST (GST-Sp110 or GST-Sp110b) or with GST as a negative control. The GST pulldown assay was performed as described in Materials and Methods. 1/10 input, signal for 1/10 the amount of 35 S-labeled product used in the pulldown assay. Coomassie brilliant blue (CBB) staining patterns of pulled-down proteins are shown in the bottom panel. Arrowhead, circle, and square indicate the bands corresponding to GST, GST-Sp110, and GST-Sp110b, respectively. (B) Mapping of the region interacting with the core in Sp110b by deletion analysis. (Left) Schematic representations of the full-length and truncated mutants of Sp110 and Sp110b are shown. Numbers above the diagrams indicate the amino acid positions from the amino terminus of Sp110 or Sp110b. The Sp100-like domain (Sp), SAND domain (S), PHD (P), and bromodomain (B) are indicated. (Right) Designations of the GST fusion protein and 35 S-labeled derivatives of Sp110 and Sp110b are given above and to the left of the autoradiograms (a through k), respectively. GST-core80, GST fused with the region of the core from aa 1 to 80. CBB staining patterns of pulled-down proteins are shown in the bottom panel. Arrow and arrowhead indicate bands corresponding to GST and GST-core80, respectively. Two dots indicate apparent degradation products of GST-core80. (C) Interaction between the core and Sp110b produced in the cells. Lysates from COS-7 cells transfected with 1 μg of pCMV-Sp110b and/or pCMV-core were used for coimmunoprecipitation, followed by immunoblot analysis. The combinations of plasmids used for the transfection are indicated at the top. “IP” designates the antibodies used for immunoprecipitation, either the anti-FLAG antibody (FL) or normal mouse IgG (used as a negative control). Coimmunoprecipitated core with FLAG-tagged Sp110b was detected with an anti-core antibody (top panel). Center and bottom panels show results of experiments in which the core and FLAG-tagged Sp110b in total-cell lysates from transfectants were detected by anti-core and anti-FLAG antibodies, respectively.
Figure Legend Snippet: Interaction of the core with Sp110b. (A) The 35 S-labeled in vitro transcription-translation product of full-length core (top panel) or the core(6162M) mutant (center panel) was incubated with recombinant Sp110 or Sp110b fused to GST (GST-Sp110 or GST-Sp110b) or with GST as a negative control. The GST pulldown assay was performed as described in Materials and Methods. 1/10 input, signal for 1/10 the amount of 35 S-labeled product used in the pulldown assay. Coomassie brilliant blue (CBB) staining patterns of pulled-down proteins are shown in the bottom panel. Arrowhead, circle, and square indicate the bands corresponding to GST, GST-Sp110, and GST-Sp110b, respectively. (B) Mapping of the region interacting with the core in Sp110b by deletion analysis. (Left) Schematic representations of the full-length and truncated mutants of Sp110 and Sp110b are shown. Numbers above the diagrams indicate the amino acid positions from the amino terminus of Sp110 or Sp110b. The Sp100-like domain (Sp), SAND domain (S), PHD (P), and bromodomain (B) are indicated. (Right) Designations of the GST fusion protein and 35 S-labeled derivatives of Sp110 and Sp110b are given above and to the left of the autoradiograms (a through k), respectively. GST-core80, GST fused with the region of the core from aa 1 to 80. CBB staining patterns of pulled-down proteins are shown in the bottom panel. Arrow and arrowhead indicate bands corresponding to GST and GST-core80, respectively. Two dots indicate apparent degradation products of GST-core80. (C) Interaction between the core and Sp110b produced in the cells. Lysates from COS-7 cells transfected with 1 μg of pCMV-Sp110b and/or pCMV-core were used for coimmunoprecipitation, followed by immunoblot analysis. The combinations of plasmids used for the transfection are indicated at the top. “IP” designates the antibodies used for immunoprecipitation, either the anti-FLAG antibody (FL) or normal mouse IgG (used as a negative control). Coimmunoprecipitated core with FLAG-tagged Sp110b was detected with an anti-core antibody (top panel). Center and bottom panels show results of experiments in which the core and FLAG-tagged Sp110b in total-cell lysates from transfectants were detected by anti-core and anti-FLAG antibodies, respectively.

Techniques Used: Labeling, In Vitro, Mutagenesis, Incubation, Recombinant, Negative Control, GST Pulldown Assay, Staining, Produced, Transfection, Immunoprecipitation

20) Product Images from "Renalase is a novel, soluble monoamine oxidase that regulates cardiac function and blood pressure"

Article Title: Renalase is a novel, soluble monoamine oxidase that regulates cardiac function and blood pressure

Journal:

doi: 10.1172/JCI200524066

Renalase is a secreted amine oxidase. ( A ) Detection of renalase in culture medium of HEK293 cells transiently transfected with renalase cDNA. Control, secondary antibody alone. ( B ) Western blot analysis of human plasma using an anti-renalase antibody.
Figure Legend Snippet: Renalase is a secreted amine oxidase. ( A ) Detection of renalase in culture medium of HEK293 cells transiently transfected with renalase cDNA. Control, secondary antibody alone. ( B ) Western blot analysis of human plasma using an anti-renalase antibody.

Techniques Used: Transfection, Western Blot

Renalase degrades catecholamines in vitro and regulates systemic blood pressure in vivo.
Figure Legend Snippet: Renalase degrades catecholamines in vitro and regulates systemic blood pressure in vivo.

Techniques Used: In Vitro, In Vivo

Tissue expression of renalase. ( A ) Northern blot analysis of human tissues using the MGC12474 clone as a probe. The upper band in skeletal muscle and lower bands in kidney and liver may represent alternatively spliced forms of renalase. ( B ) Western blot
Figure Legend Snippet: Tissue expression of renalase. ( A ) Northern blot analysis of human tissues using the MGC12474 clone as a probe. The upper band in skeletal muscle and lower bands in kidney and liver may represent alternatively spliced forms of renalase. ( B ) Western blot

Techniques Used: Expressing, Northern Blot, Western Blot

Effect on renalase of hemodynamic parameters. ( A ) Cardiac response before and after an i.v. bolus injection of 4 μg/g body wt. The arrow denotes the timing of renalase injection. VP, left ventricular pressure; VP max, maximal left ventricular
Figure Legend Snippet: Effect on renalase of hemodynamic parameters. ( A ) Cardiac response before and after an i.v. bolus injection of 4 μg/g body wt. The arrow denotes the timing of renalase injection. VP, left ventricular pressure; VP max, maximal left ventricular

Techniques Used: Injection

21) Product Images from "Dual roles of the SUMO-interacting motif in the regulation of Srs2 sumoylation"

Article Title: Dual roles of the SUMO-interacting motif in the regulation of Srs2 sumoylation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks484

SUMO-PCNA inhibits Srs2 sumoylation by binding to the SIM of Srs2. ( A ) SUMO-PCNA inhibits Srs2 sumoylation in vitro . In vitro sumoylation assay was performed using Aos1/Uba2 (0.35 μM), Ubc9 (1.25 μM), SUMO (1.6 μM), Siz1 (1–465) (0.4 μM), Srs2 (0.75 μM) and ATP (100 μM) in the absence or presence of increasing amounts of SUMO-PCNA (0.8, 1.6, 3.2 μM, lanes 2–4) or PCNA-K164R (lanes 8–10). The reactions were stopped, resolved on 10% SDS-PAGE gel and stained with Coomassie Blue. ( B ) SUMO-PCNA outcompetes SUMO in Srs2 binding. Pull-down experiments using purified GST-SUMO (2 μM, lanes 1–4) and Srs2 (0.6 μM) in the absence (lanes 1 and 2) or presence of SUMO-PCNA (0.6 μM, lanes 3–6) were performed as in Figure 3 . ( C ) Lack of PCNA sumoylation alters the Srs2 sumoylation profile in vivo . Endogenous Srs2 from wild-type or pol30-K127, 164R yeast strains treated with 0.3% MMS was analyzed as in Figure 2 B. ( D ) The inhibitory effect of SUMO-PCNA on Srs2 sumoylation can be overcome by increasing amounts of SUMO and Siz1. Srs2 (0.75 μM) was pre-incubated with SUMO-PCNA (1.5 μM, lanes 1–4) for 15 min at RT, after which the sumoylation assay was performed using Aos1/Uba2 (0.35 μM), indicated amounts of Ubc9, SUMO and Siz1(1–465) in buffer containing 100 μM ATP and 300 mM KCl. Reactions were stopped and analyzed by 10% SDS-PAGE, followed by Coomassie Blue staining.
Figure Legend Snippet: SUMO-PCNA inhibits Srs2 sumoylation by binding to the SIM of Srs2. ( A ) SUMO-PCNA inhibits Srs2 sumoylation in vitro . In vitro sumoylation assay was performed using Aos1/Uba2 (0.35 μM), Ubc9 (1.25 μM), SUMO (1.6 μM), Siz1 (1–465) (0.4 μM), Srs2 (0.75 μM) and ATP (100 μM) in the absence or presence of increasing amounts of SUMO-PCNA (0.8, 1.6, 3.2 μM, lanes 2–4) or PCNA-K164R (lanes 8–10). The reactions were stopped, resolved on 10% SDS-PAGE gel and stained with Coomassie Blue. ( B ) SUMO-PCNA outcompetes SUMO in Srs2 binding. Pull-down experiments using purified GST-SUMO (2 μM, lanes 1–4) and Srs2 (0.6 μM) in the absence (lanes 1 and 2) or presence of SUMO-PCNA (0.6 μM, lanes 3–6) were performed as in Figure 3 . ( C ) Lack of PCNA sumoylation alters the Srs2 sumoylation profile in vivo . Endogenous Srs2 from wild-type or pol30-K127, 164R yeast strains treated with 0.3% MMS was analyzed as in Figure 2 B. ( D ) The inhibitory effect of SUMO-PCNA on Srs2 sumoylation can be overcome by increasing amounts of SUMO and Siz1. Srs2 (0.75 μM) was pre-incubated with SUMO-PCNA (1.5 μM, lanes 1–4) for 15 min at RT, after which the sumoylation assay was performed using Aos1/Uba2 (0.35 μM), indicated amounts of Ubc9, SUMO and Siz1(1–465) in buffer containing 100 μM ATP and 300 mM KCl. Reactions were stopped and analyzed by 10% SDS-PAGE, followed by Coomassie Blue staining.

Techniques Used: Binding Assay, In Vitro, SDS Page, Staining, Purification, In Vivo, Incubation

The interaction between the Srs2 SIM motif and SUMO is necessary for Srs2 sumoylation. ( A ) SUMO interacts with the Srs2 SIM motif. Purified GST-SUMO (2 μM, lanes 1, 2 and 5, 6) was incubated with Srs2 (0.6 μM, lanes 1–4) or its mutant lacking the SIM motif—Srs2ΔSIM (0.6 μM, lanes 5–8) and GTH-Sepharose in buffer S2 containing 100 mM KCl for 30 min at RT. The beads were washed and treated with SDS Laemmli buffer to elute the bound proteins. The supernatant (S) containing unbound Srs2 protein, and the SDS eluate (E) (10 μl each) were analyzed by 10% SDS–PAGE followed by staining with Coomassie Blue. Reactions containing only GTH-Sepharose and Srs2 (lanes 3 and 4) or Srs2ΔSIM (lanes 7 and 8) were included as negative control. Numbers on the left side of the gel indicate molecular weights (in kDa) of protein standards. ( B ) Ubc9 does not interact with Srs2. Interaction between purified GST-Ubc9 (4 μM, lanes 1–3) and Srs2 (1.25 μM) was analyzed as in (A). ( C ) SUMO modification of Ubc9 triggers its interaction with Srs2. GST-Ubc9 (4 μM, lanes 1–2) or GST-Ubc9-SUMO (4 μM, lanes 3–6), prepared by sumoylation reaction in the absence or presence of ATP, was mixed with Srs2 (1.25 μM, lanes 1–4) or Srs2ΔSIM (1.25 μM, lanes 5–6) and analyzed as in (A), except β-mercaptoethanol was excluded from the Laemmli buffer to prevent denaturation of SUMO-charged Ubc9. ( D ) Yeast two-hybrid interaction of Ubc9 and SUMO with Srs2 is mediated by its SIM motif. Strain PJ69-4 containing UBC9 or SUMO fused to the GAL4 transcription activation domain and SRS2 (aa 783–1174) or SRS2ΔSIM (a.a. 783–1169) fused to the GAL4 DNA-binding domain, were spotted as 10-fold serial dilutions on medium lacking leucine and tryptophan or leucine, tryptophan and histidine. The empty vector (pGADT7) was included as negative control. ( E ) Srs2 SIM motif is necessary for Srs2 sumoylation in vitro . The standard in vitro sumoylation reaction was done with wild-type Srs2 (lanes 1 and 2) or Srs2ΔSIM (lanes 3 and 4) in buffer S2 containing 100 mM KCl. ( F ) In vivo sumoylation of Srs2 requires its SIM motif. Yeast cells, expressing His-tagged wild-type Srs2 or Srs2ΔSIM mutant under the copper-responsive CUP1 promoter, were grown in the absence or presence of 0.3% MMS and immunoprecipitated using anti-Srs2 antibody. Western blotting was performed as in Figure 2 B.
Figure Legend Snippet: The interaction between the Srs2 SIM motif and SUMO is necessary for Srs2 sumoylation. ( A ) SUMO interacts with the Srs2 SIM motif. Purified GST-SUMO (2 μM, lanes 1, 2 and 5, 6) was incubated with Srs2 (0.6 μM, lanes 1–4) or its mutant lacking the SIM motif—Srs2ΔSIM (0.6 μM, lanes 5–8) and GTH-Sepharose in buffer S2 containing 100 mM KCl for 30 min at RT. The beads were washed and treated with SDS Laemmli buffer to elute the bound proteins. The supernatant (S) containing unbound Srs2 protein, and the SDS eluate (E) (10 μl each) were analyzed by 10% SDS–PAGE followed by staining with Coomassie Blue. Reactions containing only GTH-Sepharose and Srs2 (lanes 3 and 4) or Srs2ΔSIM (lanes 7 and 8) were included as negative control. Numbers on the left side of the gel indicate molecular weights (in kDa) of protein standards. ( B ) Ubc9 does not interact with Srs2. Interaction between purified GST-Ubc9 (4 μM, lanes 1–3) and Srs2 (1.25 μM) was analyzed as in (A). ( C ) SUMO modification of Ubc9 triggers its interaction with Srs2. GST-Ubc9 (4 μM, lanes 1–2) or GST-Ubc9-SUMO (4 μM, lanes 3–6), prepared by sumoylation reaction in the absence or presence of ATP, was mixed with Srs2 (1.25 μM, lanes 1–4) or Srs2ΔSIM (1.25 μM, lanes 5–6) and analyzed as in (A), except β-mercaptoethanol was excluded from the Laemmli buffer to prevent denaturation of SUMO-charged Ubc9. ( D ) Yeast two-hybrid interaction of Ubc9 and SUMO with Srs2 is mediated by its SIM motif. Strain PJ69-4 containing UBC9 or SUMO fused to the GAL4 transcription activation domain and SRS2 (aa 783–1174) or SRS2ΔSIM (a.a. 783–1169) fused to the GAL4 DNA-binding domain, were spotted as 10-fold serial dilutions on medium lacking leucine and tryptophan or leucine, tryptophan and histidine. The empty vector (pGADT7) was included as negative control. ( E ) Srs2 SIM motif is necessary for Srs2 sumoylation in vitro . The standard in vitro sumoylation reaction was done with wild-type Srs2 (lanes 1 and 2) or Srs2ΔSIM (lanes 3 and 4) in buffer S2 containing 100 mM KCl. ( F ) In vivo sumoylation of Srs2 requires its SIM motif. Yeast cells, expressing His-tagged wild-type Srs2 or Srs2ΔSIM mutant under the copper-responsive CUP1 promoter, were grown in the absence or presence of 0.3% MMS and immunoprecipitated using anti-Srs2 antibody. Western blotting was performed as in Figure 2 B.

Techniques Used: Purification, Incubation, Mutagenesis, SDS Page, Staining, Negative Control, Modification, Activation Assay, Binding Assay, Plasmid Preparation, In Vitro, In Vivo, Expressing, Immunoprecipitation, Western Blot

Sumoylation of Srs2 inhibits its interaction with SUMO-PCNA and SUMO in vitro . ( A ) Sumoylation of Srs2 inhibits its interaction with SUMO-PCNA. His-tagged Srs2 (1.5 μM, lanes 1 and 2) or SUMO-Srs2 (1.5 μM, lanes 3 and 4), prepared by sumoylation reaction using untagged SUMO and Siz1 proteins, was mixed with SUMO-PCNA (1.5 μM) and Ni-charged resin. The beads were washed and treated with SDS Laemmli buffer to elute the bound proteins. The supernatant (S) containing unbound Srs2 protein and the SDS eluate (E) (10 μl each) were analyzed by 10% SDS–PAGE followed by Coomassie Blue staining. Sumoylation reaction in the absence of Srs2 was mixed with Ni-charged resin as a negative control (lanes 5 and 6). ( B ) Sumoylation of Srs2 inhibits its interaction with SUMO. Srs2 (1.5 μM, lanes 1–4) or SUMO-Srs2 (1.5 μM, lanes 5–8), prepared by sumoylation reaction in the absence or presence of ATP, was mixed with GST-SUMO (1.5 μM, lanes 1, 2, 5 and 6) or without it (lanes 3, 4, 7 and 8) and pulled-down on GTH-Sepharose beads. The analysis was performed as in (A).
Figure Legend Snippet: Sumoylation of Srs2 inhibits its interaction with SUMO-PCNA and SUMO in vitro . ( A ) Sumoylation of Srs2 inhibits its interaction with SUMO-PCNA. His-tagged Srs2 (1.5 μM, lanes 1 and 2) or SUMO-Srs2 (1.5 μM, lanes 3 and 4), prepared by sumoylation reaction using untagged SUMO and Siz1 proteins, was mixed with SUMO-PCNA (1.5 μM) and Ni-charged resin. The beads were washed and treated with SDS Laemmli buffer to elute the bound proteins. The supernatant (S) containing unbound Srs2 protein and the SDS eluate (E) (10 μl each) were analyzed by 10% SDS–PAGE followed by Coomassie Blue staining. Sumoylation reaction in the absence of Srs2 was mixed with Ni-charged resin as a negative control (lanes 5 and 6). ( B ) Sumoylation of Srs2 inhibits its interaction with SUMO. Srs2 (1.5 μM, lanes 1–4) or SUMO-Srs2 (1.5 μM, lanes 5–8), prepared by sumoylation reaction in the absence or presence of ATP, was mixed with GST-SUMO (1.5 μM, lanes 1, 2, 5 and 6) or without it (lanes 3, 4, 7 and 8) and pulled-down on GTH-Sepharose beads. The analysis was performed as in (A).

Techniques Used: In Vitro, SDS Page, Staining, Negative Control

22) Product Images from "Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes"

Article Title: Interaction of two photoreceptors in the regulation of bacterial photosynthesis genes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks243

In vitro interaction of CryB and AppA. Western blots of 12% SDS–PAGE from [glutathione S transferase (GST)- and MBP-] pull-down assays using a CryB-specific antibody (A–D) or a LOV-specific antibody (E). ( A ) AppA-MBP protein bound to amylose–agarose and incubated with cell lysate from R.s. Δ cryB (pRK pufcryB ). ( B ) Incubation of cell lysate from R.s. Δ cryB (pRK pufcryB ) with amylose–agarose. ( C ) GST-AppAΔN bound to glutathione-sepharose and incubated with cell lysate from R.s. Δ cryB (pRK pufcryB ). ( D ) GST-SCHIC bound to glutathione–sepharose and incubated with cell lysate from R.s. Δ cryB (pRK pufcryB ). ( E ) AppA-MBP protein bound to amylose–agarose and incubated with cell lysate from R.s. 2.4.1(pRK puflov ). F, cell lysate flow through; W, washing fractions (same volume as F); E, elution fractions (same volume as F).
Figure Legend Snippet: In vitro interaction of CryB and AppA. Western blots of 12% SDS–PAGE from [glutathione S transferase (GST)- and MBP-] pull-down assays using a CryB-specific antibody (A–D) or a LOV-specific antibody (E). ( A ) AppA-MBP protein bound to amylose–agarose and incubated with cell lysate from R.s. Δ cryB (pRK pufcryB ). ( B ) Incubation of cell lysate from R.s. Δ cryB (pRK pufcryB ) with amylose–agarose. ( C ) GST-AppAΔN bound to glutathione-sepharose and incubated with cell lysate from R.s. Δ cryB (pRK pufcryB ). ( D ) GST-SCHIC bound to glutathione–sepharose and incubated with cell lysate from R.s. Δ cryB (pRK pufcryB ). ( E ) AppA-MBP protein bound to amylose–agarose and incubated with cell lysate from R.s. 2.4.1(pRK puflov ). F, cell lysate flow through; W, washing fractions (same volume as F); E, elution fractions (same volume as F).

Techniques Used: In Vitro, Western Blot, SDS Page, Incubation, Flow Cytometry

23) Product Images from "MPP6 is an exosome-associated RNA-binding protein involved in 5.8S rRNA maturation"

Article Title: MPP6 is an exosome-associated RNA-binding protein involved in 5.8S rRNA maturation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki982

Knock down of MPP6 by RNAi leads to the accumulation of 5.8S rRNA precursors. ( A ) HEp-2 cells were transiently transfected with three different siRNAs to MPP6 (100 pmol), control siRNA or buffer (mock). Cells were harvested 2 days after transfection and 75 µg of total protein was analysed by western blotting using a polyclonal anti-MPP6 serum or a polyclonal anti-hRrp41p serum (control). ( B ) Northern analysis of 5.8S rRNA processing upon MPP6 knock down. Total RNA (5 µg) from (mock) transfected cells was analysed by northern blot hybridization using radiolabelled probes specific for 5.8S rRNA (left) or ITS2 (right). The relative positions of these probes, and also the other probes used, with respect to the primary rRNA transcript are depicted in the scheme below the autoradiographs. Note that the size of the probes is not proportional to that of the pre-rRNA. As a control, a U6 snRNA probe was used. The positions of 5.8S rRNA and its precursors (I and II) are indicated. The asterisk in the right panel points to a weak cross-hybridization of the ITS2 probe with the mature 5.8S rRNA.
Figure Legend Snippet: Knock down of MPP6 by RNAi leads to the accumulation of 5.8S rRNA precursors. ( A ) HEp-2 cells were transiently transfected with three different siRNAs to MPP6 (100 pmol), control siRNA or buffer (mock). Cells were harvested 2 days after transfection and 75 µg of total protein was analysed by western blotting using a polyclonal anti-MPP6 serum or a polyclonal anti-hRrp41p serum (control). ( B ) Northern analysis of 5.8S rRNA processing upon MPP6 knock down. Total RNA (5 µg) from (mock) transfected cells was analysed by northern blot hybridization using radiolabelled probes specific for 5.8S rRNA (left) or ITS2 (right). The relative positions of these probes, and also the other probes used, with respect to the primary rRNA transcript are depicted in the scheme below the autoradiographs. Note that the size of the probes is not proportional to that of the pre-rRNA. As a control, a U6 snRNA probe was used. The positions of 5.8S rRNA and its precursors (I and II) are indicated. The asterisk in the right panel points to a weak cross-hybridization of the ITS2 probe with the mature 5.8S rRNA.

Techniques Used: Transfection, Western Blot, Northern Blot, Hybridization

MPP6, hRrp41 and PM/Scl-100 knock down lead to the accumulation of the same 5.8S rRNA precursors. ( A ) HEp-2 cells were transiently transfected with control siRNA (100 pmol), MPP6-2 siRNA (100 pmol), hRrp41 siRNA (200 pmol) and PM/Scl-100 siRNA (100 pmol). After 36 h transfection cells were retransfected with the same amount of siRNA and after an additional 36 h the cells were harvested. The proteins were analysed by western blotting using 75 µg of total protein extracts from transfected cells and a polyclonal anti-MPP6 serum, a polyclonal anti-hRrp41 serum, and a polyclonal anti-PM/Scl-100 serum. A polyclonal anti-hRpp40 serum was used as a control. ( B ) A radiolabelled antisense ITS2 probe, complementary to the first 300 nt of ITS2, was hybridized to 5 µg of total RNA from siRNA-treated cells. After digestion with RNase A and RNase T1 the RNA was analysed on a 10% denaturing polyacrylamide gel. The radiolabelled ITS2 probe was loaded in lane 1. As a control, the same probe was incubated with 5 µg of yeast total RNA and further treated by the same procedure (lane 6). Lanes 4–6 show protected RNAs from cells treated with control, MPP6-2, Rrp41 and PM/Scl-100 siRNA, respectively. The positions of the 5.8S precursor rRNAs (I and II) are indicated. Positions of marker RNAs are indicated on the left.
Figure Legend Snippet: MPP6, hRrp41 and PM/Scl-100 knock down lead to the accumulation of the same 5.8S rRNA precursors. ( A ) HEp-2 cells were transiently transfected with control siRNA (100 pmol), MPP6-2 siRNA (100 pmol), hRrp41 siRNA (200 pmol) and PM/Scl-100 siRNA (100 pmol). After 36 h transfection cells were retransfected with the same amount of siRNA and after an additional 36 h the cells were harvested. The proteins were analysed by western blotting using 75 µg of total protein extracts from transfected cells and a polyclonal anti-MPP6 serum, a polyclonal anti-hRrp41 serum, and a polyclonal anti-PM/Scl-100 serum. A polyclonal anti-hRpp40 serum was used as a control. ( B ) A radiolabelled antisense ITS2 probe, complementary to the first 300 nt of ITS2, was hybridized to 5 µg of total RNA from siRNA-treated cells. After digestion with RNase A and RNase T1 the RNA was analysed on a 10% denaturing polyacrylamide gel. The radiolabelled ITS2 probe was loaded in lane 1. As a control, the same probe was incubated with 5 µg of yeast total RNA and further treated by the same procedure (lane 6). Lanes 4–6 show protected RNAs from cells treated with control, MPP6-2, Rrp41 and PM/Scl-100 siRNA, respectively. The positions of the 5.8S precursor rRNAs (I and II) are indicated. Positions of marker RNAs are indicated on the left.

Techniques Used: Transfection, Western Blot, Incubation, Marker

MPP6 is associated with nuclear exosome complexes. ( A ) HEp-2 cell extracts were subjected to immunoprecipitation with three anti-PM/Scl-positive (lanes 1–3) and three anti-PM/Scl-negative patient sera (lanes 4–6). Precipitated proteins were analysed by western blotting using a rabbit anti-MPP6 serum. The positions of molecular mass markers (kDa) are indicated on the left. ( B ) Reciprocal experiment in which a normal rabbit serum (lanes 2, 6 and 10), a polyclonal anti-MPP6 serum (lanes 3, 7 and 11) and a polyclonal anti-hRrp40p serum (lanes 4, 8 and 12) were used for immunoprecipitations. A monoclonal antibody to hRrp4p was used for the detection of a co-precipitated exosome component. For the immunoprecipitations total (lanes 1–4), nuclear (lanes 5–8) and cytoplasmic (lanes 9–12) HEp-2 cell extracts were used. Input material (10%) of the extracts was loaded in lanes 1, 5 and 9, respectively. The positions of molecular mass markers (kDa) are indicated on the left.
Figure Legend Snippet: MPP6 is associated with nuclear exosome complexes. ( A ) HEp-2 cell extracts were subjected to immunoprecipitation with three anti-PM/Scl-positive (lanes 1–3) and three anti-PM/Scl-negative patient sera (lanes 4–6). Precipitated proteins were analysed by western blotting using a rabbit anti-MPP6 serum. The positions of molecular mass markers (kDa) are indicated on the left. ( B ) Reciprocal experiment in which a normal rabbit serum (lanes 2, 6 and 10), a polyclonal anti-MPP6 serum (lanes 3, 7 and 11) and a polyclonal anti-hRrp40p serum (lanes 4, 8 and 12) were used for immunoprecipitations. A monoclonal antibody to hRrp4p was used for the detection of a co-precipitated exosome component. For the immunoprecipitations total (lanes 1–4), nuclear (lanes 5–8) and cytoplasmic (lanes 9–12) HEp-2 cell extracts were used. Input material (10%) of the extracts was loaded in lanes 1, 5 and 9, respectively. The positions of molecular mass markers (kDa) are indicated on the left.

Techniques Used: Immunoprecipitation, Western Blot

Model for the role of MPP6 in pre-rRNA processing. Before, or directly after cleavage of the pre-rRNA in ITS2 by a yet unknown endoribonuclease, MPP6 binds to oligopyrimidine stretches in the ITS2 RNA and subsequently recruits the PM/Scl-100-containing exosome and probably several additional factors, like hMtr4p or an hMtr4p containing complex. This is in agreement with the reported two-hybrid interactions between MPP6 and PM/Scl-100 and between MPP6 and hMtr4p ( 25 ). The exosome will then process the ITS2 RNA to generate the mature 5.8S rRNA. MPP6 may either remain associated with the processing complex or dissociate once the exosome starts the digestion.
Figure Legend Snippet: Model for the role of MPP6 in pre-rRNA processing. Before, or directly after cleavage of the pre-rRNA in ITS2 by a yet unknown endoribonuclease, MPP6 binds to oligopyrimidine stretches in the ITS2 RNA and subsequently recruits the PM/Scl-100-containing exosome and probably several additional factors, like hMtr4p or an hMtr4p containing complex. This is in agreement with the reported two-hybrid interactions between MPP6 and PM/Scl-100 and between MPP6 and hMtr4p ( 25 ). The exosome will then process the ITS2 RNA to generate the mature 5.8S rRNA. MPP6 may either remain associated with the processing complex or dissociate once the exosome starts the digestion.

Techniques Used:

MPP6 predominantly co-sediments with nuclear exosome complexes at 60S–80S in glycerol gradients. Cytoplasmic ( A ) and nuclear ( B ) extracts from HEp-2 cells were fractionated in a 5–40% (v/v) glycerol gradient. The sedimentation of hRrp4, MPP6 and PM/Scl-100 was determined by immunoblotting. The sedimentation of the large rRNAs was determined by agarose gel electrophoresis and ethidium bromide staining and used as markers (40S and 60S) in the gradient. U1 snRNA was used as marker for 12S complexes.
Figure Legend Snippet: MPP6 predominantly co-sediments with nuclear exosome complexes at 60S–80S in glycerol gradients. Cytoplasmic ( A ) and nuclear ( B ) extracts from HEp-2 cells were fractionated in a 5–40% (v/v) glycerol gradient. The sedimentation of hRrp4, MPP6 and PM/Scl-100 was determined by immunoblotting. The sedimentation of the large rRNAs was determined by agarose gel electrophoresis and ethidium bromide staining and used as markers (40S and 60S) in the gradient. U1 snRNA was used as marker for 12S complexes.

Techniques Used: Sedimentation, Agarose Gel Electrophoresis, Staining, Marker

Subcellular localization of MPP6. HEp-2 cells were transfected with constructs encoding EGFP alone ( A and D ), EGFP-MPP6 ( B and E ) and EGFP-hMtr3p ( C and F ) and after 24 h the cells were fixed in 4% paraformaldehyde/PBS and the expressed fusion proteins were analysed by fluorescence microscopy. Phase-contrast and fluorescence images are displayed in (A–C and D–F), respectively. Each bar corresponds to 10 µm.
Figure Legend Snippet: Subcellular localization of MPP6. HEp-2 cells were transfected with constructs encoding EGFP alone ( A and D ), EGFP-MPP6 ( B and E ) and EGFP-hMtr3p ( C and F ) and after 24 h the cells were fixed in 4% paraformaldehyde/PBS and the expressed fusion proteins were analysed by fluorescence microscopy. Phase-contrast and fluorescence images are displayed in (A–C and D–F), respectively. Each bar corresponds to 10 µm.

Techniques Used: Transfection, Construct, Fluorescence, Microscopy

GST-tagged MPP6 binds to in vitro transcribed RNAs and prefers polypyrimidines. ( A ) GST and GST-MPP6 recombinant proteins were incubated with radiolabeled, in vitro transcribed full-length 5.8S rRNA, ITS2 rRNA (a fragment corresponding to the most 5′ 300 nt) and RNase MRP RNA. Binding to these RNAs was assayed by GST pull-down followed by denaturing gel electrophoresis and autoradiography. Five percent of the input RNA was loaded in lanes 1. Lanes 2 and 3 contain the material precipitated by GST alone and GST-MPP6, respectively. ( B ) Binding of GST and GST–MPP6 fusion proteins to radiolabelled homopolynucleotides was analysed as described above and the bound RNAs were quantified in a scintillation counter. The binding efficiency of GST or GST-MPP6 with poly(A), poly(C), poly(G), poly(U) and poly(C)–ply(I) is depicted as a percentage of input RNA (RBE: relative binding efficiency). These results are the averages of two independent experiments.
Figure Legend Snippet: GST-tagged MPP6 binds to in vitro transcribed RNAs and prefers polypyrimidines. ( A ) GST and GST-MPP6 recombinant proteins were incubated with radiolabeled, in vitro transcribed full-length 5.8S rRNA, ITS2 rRNA (a fragment corresponding to the most 5′ 300 nt) and RNase MRP RNA. Binding to these RNAs was assayed by GST pull-down followed by denaturing gel electrophoresis and autoradiography. Five percent of the input RNA was loaded in lanes 1. Lanes 2 and 3 contain the material precipitated by GST alone and GST-MPP6, respectively. ( B ) Binding of GST and GST–MPP6 fusion proteins to radiolabelled homopolynucleotides was analysed as described above and the bound RNAs were quantified in a scintillation counter. The binding efficiency of GST or GST-MPP6 with poly(A), poly(C), poly(G), poly(U) and poly(C)–ply(I) is depicted as a percentage of input RNA (RBE: relative binding efficiency). These results are the averages of two independent experiments.

Techniques Used: In Vitro, Recombinant, Incubation, RNA Binding Assay, Nucleic Acid Electrophoresis, Autoradiography, Binding Assay

24) Product Images from "Endocytosis of Seven-Transmembrane RGS Protein Activates G- protein Coupled Signaling in Arabidopsis"

Article Title: Endocytosis of Seven-Transmembrane RGS Protein Activates G- protein Coupled Signaling in Arabidopsis

Journal: Nature cell biology

doi: 10.1038/ncb2568

In vivo and In vitro function of AtWNK8 ( A ) In vivo phosphorylation of AtRGS1. Seedlings expressing AtRGS1-TAP were pretreated with 100 nM calyculin A and 10 mM sodium orthovanadate for 3 h followed by 6% D-glucose stimulation for 90 min. AtRGS1-TAP or AtGPA1 in seedling lysates was separated on a 12.5% Anderson’s gel and detected by immunoblot with peroxidase anti-peroxidase or anti-AtGPA1 antibody. ( B ) Four-day-old AtRGS1-YFP expressing seedlings were treated with phosphatase inhibitors, calyculin A, for 2 h followed by 6% glucose treatment or not (No glucose) for 1 h prior to imaging epidermal cells. Scale bars = 10 µm. Error = SEM, n = 5. ( C ) Phylogenetic tree of the AtWNK-family kinases. Full-length amino acid sequences were aligned with CLUSTAL W implemented in CLC Genomics Workbench using the following settings; Gap open penalty, 10; Gap extension penalty 1. The neighbor joining tree (1000 bootstrap replicate) was created with the aligned sequences. ( D ) In vitro binding between AtRGS1 and AtWNKs. Recombinant RGSbox+Cterm was tested for interaction with GST (negative control) or GST-AtWNKs using glutathione-Sepharose, and detected by immunoblot analysis using an anti-AtRGS1 antibody. ( E ) In vitro phosphorylation of AtRGS1 by AtWNK kinases. Recombinant GST or His-RGSbox+Cterm was incubated with GST-AtWNKs in reaction buffer containing γ 32 P-ATP. Proteins were separated on SDS-PAGE. ( F ) Radioactivity incorporated into the GST/RGS1 bands. Phosphorylation levels of three independent experiments were quantified in (E). Error bars = SEM. ( G ) Quantitation of sugar-induced AtRGS1 internalization in AtWNK-null mutants. Seedlings of Col-0, wnk1-1 , wnk8-1 , wnk8-2 or wnk10-2 transiently expressing AtRGS1-YFP were treated with 6% D-glucose for 30 min. WNK# denotes AtWNK members in panels C-F. Error bars = SEM, n = 5. Quantitation of fluorescence is described in Methods .
Figure Legend Snippet: In vivo and In vitro function of AtWNK8 ( A ) In vivo phosphorylation of AtRGS1. Seedlings expressing AtRGS1-TAP were pretreated with 100 nM calyculin A and 10 mM sodium orthovanadate for 3 h followed by 6% D-glucose stimulation for 90 min. AtRGS1-TAP or AtGPA1 in seedling lysates was separated on a 12.5% Anderson’s gel and detected by immunoblot with peroxidase anti-peroxidase or anti-AtGPA1 antibody. ( B ) Four-day-old AtRGS1-YFP expressing seedlings were treated with phosphatase inhibitors, calyculin A, for 2 h followed by 6% glucose treatment or not (No glucose) for 1 h prior to imaging epidermal cells. Scale bars = 10 µm. Error = SEM, n = 5. ( C ) Phylogenetic tree of the AtWNK-family kinases. Full-length amino acid sequences were aligned with CLUSTAL W implemented in CLC Genomics Workbench using the following settings; Gap open penalty, 10; Gap extension penalty 1. The neighbor joining tree (1000 bootstrap replicate) was created with the aligned sequences. ( D ) In vitro binding between AtRGS1 and AtWNKs. Recombinant RGSbox+Cterm was tested for interaction with GST (negative control) or GST-AtWNKs using glutathione-Sepharose, and detected by immunoblot analysis using an anti-AtRGS1 antibody. ( E ) In vitro phosphorylation of AtRGS1 by AtWNK kinases. Recombinant GST or His-RGSbox+Cterm was incubated with GST-AtWNKs in reaction buffer containing γ 32 P-ATP. Proteins were separated on SDS-PAGE. ( F ) Radioactivity incorporated into the GST/RGS1 bands. Phosphorylation levels of three independent experiments were quantified in (E). Error bars = SEM. ( G ) Quantitation of sugar-induced AtRGS1 internalization in AtWNK-null mutants. Seedlings of Col-0, wnk1-1 , wnk8-1 , wnk8-2 or wnk10-2 transiently expressing AtRGS1-YFP were treated with 6% D-glucose for 30 min. WNK# denotes AtWNK members in panels C-F. Error bars = SEM, n = 5. Quantitation of fluorescence is described in Methods .

Techniques Used: In Vivo, In Vitro, Expressing, Imaging, Binding Assay, Recombinant, Negative Control, Incubation, SDS Page, Radioactivity, Quantitation Assay, Fluorescence

25) Product Images from "Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients"

Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients

Journal: Journal of Biomedical Science

doi: 10.1186/1423-0127-20-27

Recognition of hnRNP DL and CNBP by anti-Sm positive SLE patient sera. GST-fused hnRNP DL and CNBP were prepared as described in Methods. The GST-fused proteins or GST were applied to SDS-PAGE and the immunoblots detected by the autosera from patient (pt) 2 and 18 are shown.
Figure Legend Snippet: Recognition of hnRNP DL and CNBP by anti-Sm positive SLE patient sera. GST-fused hnRNP DL and CNBP were prepared as described in Methods. The GST-fused proteins or GST were applied to SDS-PAGE and the immunoblots detected by the autosera from patient (pt) 2 and 18 are shown.

Techniques Used: SDS Page, Western Blot

26) Product Images from "The Shigella Type Three Secretion System Effector OspG Directly and Specifically Binds to Host Ubiquitin for Activation"

Article Title: The Shigella Type Three Secretion System Effector OspG Directly and Specifically Binds to Host Ubiquitin for Activation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0057558

The carboxyl terminus of OspG is required for interaction with ubiquitin. ( A ) Schematic presentation of OspG truncations assayed in (B-D). The gray box donates the region that shows sequence similarity to eukaryotic serine/threonine kinase sub-domains I-VII. ( B , C and D ) Pulldown assays of the binding between ubiquitin and various OspG truncation proteins. Ni-NTA Sepharose beads coated with His6-ubiquitin were incubated with GST, GST-OspG or indicated GST-OspG truncation proteins. Proteins retained on the beads were eluted and then subjected to SDS-PAGE and Coomassie blue staining analysis. ( E ) Coimmunoprecipitation assay of OspG (WT or L190D/L191D mutant) and ubiquitin interaction in transfected HEK 293T cells. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input). ( F ) Assay of OspG and ubiquitin interaction during Shigella infection. HEK 293T cells were infected with indicated Shigella deletion/complementation strains. Lysates of infected cell were subjected to anti-Flag immunoprecipitation. Δ mxiH is a type III secretion deficient strain; pME6032 is a rescue plasmid for expressing Flag-OspG (wild-type or L190D/L191D mutant) in the bacteria. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input).
Figure Legend Snippet: The carboxyl terminus of OspG is required for interaction with ubiquitin. ( A ) Schematic presentation of OspG truncations assayed in (B-D). The gray box donates the region that shows sequence similarity to eukaryotic serine/threonine kinase sub-domains I-VII. ( B , C and D ) Pulldown assays of the binding between ubiquitin and various OspG truncation proteins. Ni-NTA Sepharose beads coated with His6-ubiquitin were incubated with GST, GST-OspG or indicated GST-OspG truncation proteins. Proteins retained on the beads were eluted and then subjected to SDS-PAGE and Coomassie blue staining analysis. ( E ) Coimmunoprecipitation assay of OspG (WT or L190D/L191D mutant) and ubiquitin interaction in transfected HEK 293T cells. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input). ( F ) Assay of OspG and ubiquitin interaction during Shigella infection. HEK 293T cells were infected with indicated Shigella deletion/complementation strains. Lysates of infected cell were subjected to anti-Flag immunoprecipitation. Δ mxiH is a type III secretion deficient strain; pME6032 is a rescue plasmid for expressing Flag-OspG (wild-type or L190D/L191D mutant) in the bacteria. Shown are immunoblots of anti-Flag immunoprecipitates (Flag IP) and total cell lysates (Input).

Techniques Used: Sequencing, Binding Assay, Incubation, SDS Page, Staining, Co-Immunoprecipitation Assay, Mutagenesis, Transfection, Western Blot, Infection, Immunoprecipitation, Plasmid Preparation, Expressing

High-affinity binding between OspG and ubiquitin conjugates, poly-ubiquitin chains and free ubiquitin. ( A ) Pulldown of ubiquitin-conjugated proteins by purified GST-OspG. Glutathione-Sepharose beads coated with GST-OspG or GST alone were incubated with lysates of intact 293T cells or MG132- treated 293T cells. Proteins retained on the beads were eluted with SDS loading buffer and separated onto12% SDS-PAGE gels. Shown on the left are anti-ubiquitin immunoblots and on the right are Coomassie blue staining of GST or GST-OspG proteins present on the beads. ( B and C ) Pulldown of OpsG by K48- or K63-linked poly-ubiquitin chains. Ni-NTA Sepharose beads coated with His6-ubiquitin chains with indicated linkages were incubated with GST or GST-OspG. Proteins retained on the beads were subjected to SDS-PAGE and anti-GST immunoblotting analysis. ( D ) Pulldown of free ubiquitin by GST-OspG. GST or GST-OspG proteins were immobilized onto Glutathione Sepharose beads and the beads were then incubated with lysates of 293T cells. The interacting proteins eluted from the beads were resolved by 4–20% gradient SDS-PAGE gel and analyzed by anti-ubiquitin immunoblotting.
Figure Legend Snippet: High-affinity binding between OspG and ubiquitin conjugates, poly-ubiquitin chains and free ubiquitin. ( A ) Pulldown of ubiquitin-conjugated proteins by purified GST-OspG. Glutathione-Sepharose beads coated with GST-OspG or GST alone were incubated with lysates of intact 293T cells or MG132- treated 293T cells. Proteins retained on the beads were eluted with SDS loading buffer and separated onto12% SDS-PAGE gels. Shown on the left are anti-ubiquitin immunoblots and on the right are Coomassie blue staining of GST or GST-OspG proteins present on the beads. ( B and C ) Pulldown of OpsG by K48- or K63-linked poly-ubiquitin chains. Ni-NTA Sepharose beads coated with His6-ubiquitin chains with indicated linkages were incubated with GST or GST-OspG. Proteins retained on the beads were subjected to SDS-PAGE and anti-GST immunoblotting analysis. ( D ) Pulldown of free ubiquitin by GST-OspG. GST or GST-OspG proteins were immobilized onto Glutathione Sepharose beads and the beads were then incubated with lysates of 293T cells. The interacting proteins eluted from the beads were resolved by 4–20% gradient SDS-PAGE gel and analyzed by anti-ubiquitin immunoblotting.

Techniques Used: Binding Assay, Purification, Incubation, SDS Page, Western Blot, Staining

27) Product Images from "U1 small nuclear RNP from Trypanosoma brucei: a minimal U1 snRNA with unusual protein components"

Article Title: U1 small nuclear RNP from Trypanosoma brucei: a minimal U1 snRNA with unusual protein components

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki548

Identification and U1 snRNP association of a novel, U1-specific protein component: TbU1-24K. ( A ) ClustalW alignment of the novel protein component of the T.brucei U1 snRNP, TbU1-24K, with two putative homologs from T.cruzi . Two conserved regions are indicated by the boxed regions; the positions with asterisks are identical. Accession numbers (Gene DB): T.cruzi 877 (Tc00.1047053503877.10), T.cruzi 455 (Tc00.1047053509455.110) and T.brucei (Tb03.27F10.160). ( B ) TbU1-24K is a U1 snRNP-specific component. Extract was prepared from a T.brucei cell line, which stably expresses TAP-tagged TbU1-24K protein, and used to affinity-purify TAP-tagged complexes. Copurifying RNAs were analyzed by northern blotting, using a mixed probe (snRNA positions indicated on the right). Lane 1, 1% of input; lane 2, all of IgG-selected and TEV-released material; M , DIG marker V (Roche). ( C ) TbU1-24K coexists with TbU1-70K and Sm proteins in the same RNP complex. TAP-tag affinity purification of TbU1-24K complexes and RNA analysis was carried out as described in (A) (lane 1, 1% input; lane 2, 10% of IgG-selected and TEV-released material). Affinity-purified complexes were then immunoprecipitated with NIS (lane 3), anti TbU1-70K (lane 4) or anti-Sm antibodies (lane 5), using 30% for each immunoprecipitation. The snRNA positions are marked on the right. The slightly slower mobility of U1 snRNA in the immunoprecipitates (lanes 4 and 5) is most likely caused by comigrating tRNA released from the blocked protein A–Sepharose beads. M , DIG marker V (Roche). ( D ) In vitro U1 snRNA binding of U1 snRNP proteins. GST derivatives of the three U1-specific proteins TbU1-24K, TbU1C and TbU1-70K (lanes 3–5) were incubated with in vitro transcribed T.brucei U1 snRNA, followed by GST pull-down and analysis of coprecipitated RNA by northern hybridization with a U1-specific probe. A total of 10% of the input material was analyzed (lane 1), and a control precipitation was carried out with GST protein (lane 2). M , DIG marker V (Roche).
Figure Legend Snippet: Identification and U1 snRNP association of a novel, U1-specific protein component: TbU1-24K. ( A ) ClustalW alignment of the novel protein component of the T.brucei U1 snRNP, TbU1-24K, with two putative homologs from T.cruzi . Two conserved regions are indicated by the boxed regions; the positions with asterisks are identical. Accession numbers (Gene DB): T.cruzi 877 (Tc00.1047053503877.10), T.cruzi 455 (Tc00.1047053509455.110) and T.brucei (Tb03.27F10.160). ( B ) TbU1-24K is a U1 snRNP-specific component. Extract was prepared from a T.brucei cell line, which stably expresses TAP-tagged TbU1-24K protein, and used to affinity-purify TAP-tagged complexes. Copurifying RNAs were analyzed by northern blotting, using a mixed probe (snRNA positions indicated on the right). Lane 1, 1% of input; lane 2, all of IgG-selected and TEV-released material; M , DIG marker V (Roche). ( C ) TbU1-24K coexists with TbU1-70K and Sm proteins in the same RNP complex. TAP-tag affinity purification of TbU1-24K complexes and RNA analysis was carried out as described in (A) (lane 1, 1% input; lane 2, 10% of IgG-selected and TEV-released material). Affinity-purified complexes were then immunoprecipitated with NIS (lane 3), anti TbU1-70K (lane 4) or anti-Sm antibodies (lane 5), using 30% for each immunoprecipitation. The snRNA positions are marked on the right. The slightly slower mobility of U1 snRNA in the immunoprecipitates (lanes 4 and 5) is most likely caused by comigrating tRNA released from the blocked protein A–Sepharose beads. M , DIG marker V (Roche). ( D ) In vitro U1 snRNA binding of U1 snRNP proteins. GST derivatives of the three U1-specific proteins TbU1-24K, TbU1C and TbU1-70K (lanes 3–5) were incubated with in vitro transcribed T.brucei U1 snRNA, followed by GST pull-down and analysis of coprecipitated RNA by northern hybridization with a U1-specific probe. A total of 10% of the input material was analyzed (lane 1), and a control precipitation was carried out with GST protein (lane 2). M , DIG marker V (Roche).

Techniques Used: Stable Transfection, Northern Blot, Marker, Affinity Purification, Immunoprecipitation, In Vitro, Binding Assay, Incubation, Hybridization

T.brucei U1C (TbU1C): a U1 snRNP-specific component binding specifically to the 5′ terminal sequence of U1 snRNA. ( A ) ClustalW alignment of the protein sequences for the newly identified U1C homologs from T.brucei , T.cruzi and L.major , in comparison with the human U1C sequence. The conserved C 2 H 2 -type Zn finger within the boxed sequence is highlighted by large-size letters; asterisks indicate absolutely conserved amino acid positions. Accession numbers (GeneDB): T.brucei (Tb10.70.5640), T.cruzi (Tc00.1047053511367.354) and L.major (LmjF21.0320); human U1C (P09234). ( B ) Extract was prepared from a T.brucei cell line, which stably expresses TAP-tagged TbU1C protein, and used to affinity-purify TAP-tagged complexes. Purification was followed by analyzing copurifying RNAs by northern blotting, using a mixed snRNA probe (snRNA positions indicated on the right). M , DIG marker V (Roche). Lane 1, 1% of input; lane 2, 10% of IgG-selected and TEV-released material. Affinity-purified complexes were then immunoprecipitated with NIS (lane 3), anti TbU1-70K (lane 4) or anti-Sm antibodies (lane 5), using 30% for each immunoprecipitation. ( C ) TbU1C protein binds specifically to the 5′ terminal sequence of U1 snRNA. GST TbU1C protein was incubated with 32 P-labeled full-length U1 snRNA (lanes 1 and 2) and various U1 snRNA derivatives: U1 Δstem–loop (lanes 3 and 4), U1 Δ5′(1–14) (lanes 5, 6), U1 Δ5′(1–30) (lanes 7 and 8), U1 5′ stem–loop (lanes 9 and 10), U1 5′(1–14) (lanes 11 and 12) or a 17mer control RNA (lanes 13 and 14). In each case, 10% of the input ( I ) and the total GST pull-down material ( P ) were analyzed.
Figure Legend Snippet: T.brucei U1C (TbU1C): a U1 snRNP-specific component binding specifically to the 5′ terminal sequence of U1 snRNA. ( A ) ClustalW alignment of the protein sequences for the newly identified U1C homologs from T.brucei , T.cruzi and L.major , in comparison with the human U1C sequence. The conserved C 2 H 2 -type Zn finger within the boxed sequence is highlighted by large-size letters; asterisks indicate absolutely conserved amino acid positions. Accession numbers (GeneDB): T.brucei (Tb10.70.5640), T.cruzi (Tc00.1047053511367.354) and L.major (LmjF21.0320); human U1C (P09234). ( B ) Extract was prepared from a T.brucei cell line, which stably expresses TAP-tagged TbU1C protein, and used to affinity-purify TAP-tagged complexes. Purification was followed by analyzing copurifying RNAs by northern blotting, using a mixed snRNA probe (snRNA positions indicated on the right). M , DIG marker V (Roche). Lane 1, 1% of input; lane 2, 10% of IgG-selected and TEV-released material. Affinity-purified complexes were then immunoprecipitated with NIS (lane 3), anti TbU1-70K (lane 4) or anti-Sm antibodies (lane 5), using 30% for each immunoprecipitation. ( C ) TbU1C protein binds specifically to the 5′ terminal sequence of U1 snRNA. GST TbU1C protein was incubated with 32 P-labeled full-length U1 snRNA (lanes 1 and 2) and various U1 snRNA derivatives: U1 Δstem–loop (lanes 3 and 4), U1 Δ5′(1–14) (lanes 5, 6), U1 Δ5′(1–30) (lanes 7 and 8), U1 5′ stem–loop (lanes 9 and 10), U1 5′(1–14) (lanes 11 and 12) or a 17mer control RNA (lanes 13 and 14). In each case, 10% of the input ( I ) and the total GST pull-down material ( P ) were analyzed.

Techniques Used: Binding Assay, Sequencing, Stable Transfection, Purification, Northern Blot, Marker, Affinity Purification, Immunoprecipitation, Incubation, Labeling

TbU1-70K is a U1 snRNP-specific protein and binds in vitro specifically to the 5′ loop sequence of U1 snRNA. ( A ) Comparison of the domain structures of T.brucei (Tb08.4A8.530) and the human U1-70K (A25707) proteins. ( B ) Western blot analysis of T.brucei U1 snRNP proteins. U1 snRNPs were affinity-purified from T.brucei extract by a 2′- O -methyl RNA antisense oligonucleotide, protein was prepared and analyzed by SDS–PAGE and western blotting, using polyclonal rabbit antibodies against TbU1-70K (U1-70K) or non-immune serum (NIS). The arrow points to the immunostained TbU1-70K band of apparent molecular weight 40 kDa. Protein markers are on the right (in kDa). ( C ) U1 snRNA is specifically coprecipitated from T.brucei extract by anti-Tb U1-70 antibodies. Immunoprecipitations were carried out from T.brucei extract, using NIS, or with antibodies against the TbU1-70K protein (U1-70K) or against the trypanosome Sm proteins (Sm). RNA was purified from the immunoprecipitates and analyzed by 3′ end labeling with [ 32 P]pCp. The positions of the SL RNA and snRNAs are marked on the right. M , 32 P-labeled pBR322/HpaII markers. ( D ) RNA from the same immunoprecipitates was also analyzed by primer extension with a U1-specific oligonucleotide. In addition, RNA from a 10% aliquot of the input was included; the positions of the primer ( p ) and the U1-specific primer-extension product (U1) are marked on the right. M , 32 P-labeled pBR322/HpaII markers. ( E ) 32 P-labeled T.brucei U1 snRNA and mutant derivatives [as indicated above the lanes; see (F)] were in vitro transcribed and incubated with GST-TbU1-70K, followed by GST pull-down. For each reaction, 10% of the input ( I ) and the total precipitated material ( P ) were analyzed. M , 32 P-labeled pBR322/HpaII markers. ( F ) Sequences and proposed secondary structures of the T.brucei U1 snRNA and its mutant derivatives. The boxed sequence in the T.brucei U1 snRNA indicates the Sm site; the two arrows indicate a potential second stem–loop. Below, the sequences of the stem–loop derivatives are given; the circled nucleotides mark the two positions in the human loop that differ from the T.brucei sequence, and the single-nucleotide mutation (A21) in the mutant human loop.
Figure Legend Snippet: TbU1-70K is a U1 snRNP-specific protein and binds in vitro specifically to the 5′ loop sequence of U1 snRNA. ( A ) Comparison of the domain structures of T.brucei (Tb08.4A8.530) and the human U1-70K (A25707) proteins. ( B ) Western blot analysis of T.brucei U1 snRNP proteins. U1 snRNPs were affinity-purified from T.brucei extract by a 2′- O -methyl RNA antisense oligonucleotide, protein was prepared and analyzed by SDS–PAGE and western blotting, using polyclonal rabbit antibodies against TbU1-70K (U1-70K) or non-immune serum (NIS). The arrow points to the immunostained TbU1-70K band of apparent molecular weight 40 kDa. Protein markers are on the right (in kDa). ( C ) U1 snRNA is specifically coprecipitated from T.brucei extract by anti-Tb U1-70 antibodies. Immunoprecipitations were carried out from T.brucei extract, using NIS, or with antibodies against the TbU1-70K protein (U1-70K) or against the trypanosome Sm proteins (Sm). RNA was purified from the immunoprecipitates and analyzed by 3′ end labeling with [ 32 P]pCp. The positions of the SL RNA and snRNAs are marked on the right. M , 32 P-labeled pBR322/HpaII markers. ( D ) RNA from the same immunoprecipitates was also analyzed by primer extension with a U1-specific oligonucleotide. In addition, RNA from a 10% aliquot of the input was included; the positions of the primer ( p ) and the U1-specific primer-extension product (U1) are marked on the right. M , 32 P-labeled pBR322/HpaII markers. ( E ) 32 P-labeled T.brucei U1 snRNA and mutant derivatives [as indicated above the lanes; see (F)] were in vitro transcribed and incubated with GST-TbU1-70K, followed by GST pull-down. For each reaction, 10% of the input ( I ) and the total precipitated material ( P ) were analyzed. M , 32 P-labeled pBR322/HpaII markers. ( F ) Sequences and proposed secondary structures of the T.brucei U1 snRNA and its mutant derivatives. The boxed sequence in the T.brucei U1 snRNA indicates the Sm site; the two arrows indicate a potential second stem–loop. Below, the sequences of the stem–loop derivatives are given; the circled nucleotides mark the two positions in the human loop that differ from the T.brucei sequence, and the single-nucleotide mutation (A21) in the mutant human loop.

Techniques Used: In Vitro, Sequencing, Western Blot, Affinity Purification, SDS Page, Molecular Weight, Purification, End Labeling, Labeling, Mutagenesis, Incubation

28) Product Images from "The microtubule affinity regulating kinase MARK4 promotes axoneme extension during early ciliogenesis"

Article Title: The microtubule affinity regulating kinase MARK4 promotes axoneme extension during early ciliogenesis

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.201206013

ODF2 overexpression rescues cilia loss after MARK4 depletion. (A and B) RPE1 cells were transiently transfected with GFP-hODF2 (A) or GFP (B) and subsequently treated with control or MARK4 siRNA for 24 h before serum withdrawal and incubation for another 24 h. Cells were stained for DNA, γ-tubulin, and acetylated tubulin. The left images show merged images. Regions within the white boxes are shown at a higher magnification on the right. (C) Percentages of ciliated cells based on acetylated tubulin as a cilia marker. UT, untransfected. (D) Efficiency of MARK4 depletion in untransfected and GFP-hODF2–expressing cells were determined by quantitative fluorescence microscopy using MARK4-specific antibodies. Boxes show the top and bottom quartiles (25–75%) with a line at the median, and whiskers extend from the minimum to the maximum of all data. (E and F) HEK293T cells were transiently transfected with the indicated constructs. Immunoprecipitations (IP) were performed using anti-FLAG agarose. (E) Interacting proteins were detected by immunoblotting. (F) Quantification of E. (G) NIH 3T3 cells and NIH 3T3 cells expressing low levels of LAP-MARK4L were serum starved for 24 h, and immunoprecipitations were performed on the lysates using anti-GFP beads and probed for endogenous ODF2 and MARK4. exo., exogenous; endo., endogenous. The asterisk indicates an unspecific band. (H) For in vitro kinase assays, purified 6His-MARK4L, either catalytic active (ca) or kinase dead (kd), was incubated with recombinant ODF2 truncations. GST was used as a negative control. Samples were subjected to SDS-PAGE followed by autoradiography ( 32 P) and Coomassie Brilliant blue staining (CBB). An aliquot of each reaction was analyzed by immunoblotting with anti-MARK4. (I) Relative phosphorylation of ODF2-F1, ODF2-F2, and ODF2-F3 normalized to protein amounts. (J) Schematic representation of ODF2 truncations. A.U., arbitrary unit. Data are means ± SD of three independent experiments. *, P
Figure Legend Snippet: ODF2 overexpression rescues cilia loss after MARK4 depletion. (A and B) RPE1 cells were transiently transfected with GFP-hODF2 (A) or GFP (B) and subsequently treated with control or MARK4 siRNA for 24 h before serum withdrawal and incubation for another 24 h. Cells were stained for DNA, γ-tubulin, and acetylated tubulin. The left images show merged images. Regions within the white boxes are shown at a higher magnification on the right. (C) Percentages of ciliated cells based on acetylated tubulin as a cilia marker. UT, untransfected. (D) Efficiency of MARK4 depletion in untransfected and GFP-hODF2–expressing cells were determined by quantitative fluorescence microscopy using MARK4-specific antibodies. Boxes show the top and bottom quartiles (25–75%) with a line at the median, and whiskers extend from the minimum to the maximum of all data. (E and F) HEK293T cells were transiently transfected with the indicated constructs. Immunoprecipitations (IP) were performed using anti-FLAG agarose. (E) Interacting proteins were detected by immunoblotting. (F) Quantification of E. (G) NIH 3T3 cells and NIH 3T3 cells expressing low levels of LAP-MARK4L were serum starved for 24 h, and immunoprecipitations were performed on the lysates using anti-GFP beads and probed for endogenous ODF2 and MARK4. exo., exogenous; endo., endogenous. The asterisk indicates an unspecific band. (H) For in vitro kinase assays, purified 6His-MARK4L, either catalytic active (ca) or kinase dead (kd), was incubated with recombinant ODF2 truncations. GST was used as a negative control. Samples were subjected to SDS-PAGE followed by autoradiography ( 32 P) and Coomassie Brilliant blue staining (CBB). An aliquot of each reaction was analyzed by immunoblotting with anti-MARK4. (I) Relative phosphorylation of ODF2-F1, ODF2-F2, and ODF2-F3 normalized to protein amounts. (J) Schematic representation of ODF2 truncations. A.U., arbitrary unit. Data are means ± SD of three independent experiments. *, P

Techniques Used: Over Expression, Transfection, Incubation, Staining, Marker, Expressing, Fluorescence, Microscopy, Construct, In Vitro, Purification, Recombinant, Negative Control, SDS Page, Autoradiography

29) Product Images from "Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics"

Article Title: Discovery of Cellular Proteins Required for the Early Steps of HCV Infection Using Integrative Genomics

Journal: PLoS ONE

doi: 10.1371/journal.pone.0060333

CD63 participates in HCV entry through a direct interaction with HCV E2. (A) Co-immunoprecipitations of CD63 and CD81 by HCV E2. Extracts of Huh 7.5.1 cells, which were infected with JC1 E2 FLAG virus (MOI 0.3) for 72 hrs, were analyzed by Western blotting to detect the indicated proteins before and after immunoprecipitations with a FLAG antibody or a control mouse antibody. Actin is a negative control. (B) GST pull-down assays with purified proteins. GST-fused CD63 EC2 (GST-CD63) and GST-fused CD81 LEL (GST-CD81) proteins were expressed in E. coli and then purified ( Methods ). FLAG-tagged HCV E2 (FLAG-E2) proteins were expressed in yeast and then purified ( Methods ). After incubating the purified FLAG-E2 proteins with GST, GST-CD81 or GST-CD63 proteins for 2 hrs at 4°C, GST, GST-fusion proteins, and their associated proteins were precipitated with GSH Sepharose 4B. The resin-bound proteins were analyzed by Western blotting with antibodies against GST or FLAG. Degraded forms of GST-CD63 and GST-CD81 (indicated by asterisks) were also precipitated by the GSH resin. (C) Effect of a polypeptide corresponding to the CD63 EC2 domain on HCV infection. JFH1 5A-Rluc virus was incubated with GST or GST-CD63 for 2 hrs at 4°C. Huh7.5.1 cells were then inoculated with the virus–polypeptide mixtures by incubating for 3 hrs at 37°C, and the cells were further cultivated for 48 hrs. Virus infectivity was monitored by measuring Renilla luciferase activities in cell extracts, and normalized to the amounts of proteins in cell extracts (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in experimental lysates to that in the control lysate (PBS) are depicted. (D) Effect of an anti-CD63 antibody (BEM-1 from Santa Cruz Biotechnologies) on HCV infection. Huh7.5.1 cells were pre-incubated with a negative control mouse IgG1, a positive control anti-CD81 antibody, or an anti-CD63 antibody at the indicated concentrations for 1 hour at 37°C, and then inoculated with JFH1 5A-Rluc virus (MOI of 0.3). The cells were cultivated for additional 48 hrs, and then Renilla luciferase activities in cell lysates were measured and normalized to the amounts of proteins in lysates (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in experimental lysates to that in the control lysate (PBS) are depicted.
Figure Legend Snippet: CD63 participates in HCV entry through a direct interaction with HCV E2. (A) Co-immunoprecipitations of CD63 and CD81 by HCV E2. Extracts of Huh 7.5.1 cells, which were infected with JC1 E2 FLAG virus (MOI 0.3) for 72 hrs, were analyzed by Western blotting to detect the indicated proteins before and after immunoprecipitations with a FLAG antibody or a control mouse antibody. Actin is a negative control. (B) GST pull-down assays with purified proteins. GST-fused CD63 EC2 (GST-CD63) and GST-fused CD81 LEL (GST-CD81) proteins were expressed in E. coli and then purified ( Methods ). FLAG-tagged HCV E2 (FLAG-E2) proteins were expressed in yeast and then purified ( Methods ). After incubating the purified FLAG-E2 proteins with GST, GST-CD81 or GST-CD63 proteins for 2 hrs at 4°C, GST, GST-fusion proteins, and their associated proteins were precipitated with GSH Sepharose 4B. The resin-bound proteins were analyzed by Western blotting with antibodies against GST or FLAG. Degraded forms of GST-CD63 and GST-CD81 (indicated by asterisks) were also precipitated by the GSH resin. (C) Effect of a polypeptide corresponding to the CD63 EC2 domain on HCV infection. JFH1 5A-Rluc virus was incubated with GST or GST-CD63 for 2 hrs at 4°C. Huh7.5.1 cells were then inoculated with the virus–polypeptide mixtures by incubating for 3 hrs at 37°C, and the cells were further cultivated for 48 hrs. Virus infectivity was monitored by measuring Renilla luciferase activities in cell extracts, and normalized to the amounts of proteins in cell extracts (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in experimental lysates to that in the control lysate (PBS) are depicted. (D) Effect of an anti-CD63 antibody (BEM-1 from Santa Cruz Biotechnologies) on HCV infection. Huh7.5.1 cells were pre-incubated with a negative control mouse IgG1, a positive control anti-CD81 antibody, or an anti-CD63 antibody at the indicated concentrations for 1 hour at 37°C, and then inoculated with JFH1 5A-Rluc virus (MOI of 0.3). The cells were cultivated for additional 48 hrs, and then Renilla luciferase activities in cell lysates were measured and normalized to the amounts of proteins in lysates (mean ± s.d. from three independent experiments performed in duplicate). The relative luciferase activities in experimental lysates to that in the control lysate (PBS) are depicted.

Techniques Used: Infection, Western Blot, Negative Control, Purification, Incubation, Luciferase, Positive Control

30) Product Images from "Possible involvement of NEDD4 in keloid formation; its critical role in fibroblast proliferation and collagen production"

Article Title: Possible involvement of NEDD4 in keloid formation; its critical role in fibroblast proliferation and collagen production

Journal: Proceedings of the Japan Academy. Series B, Physical and Biological Sciences

doi: 10.2183/pjab.87.563

NEDD4 activated Akt signaling pathway through diminished PTEN protein level in fibroblasts. (A) In vitro ubiquitination assay of PTEN by NEDD4. NEDD4 ubiquitinated PTEN directly. (B) NEDD4 ubiquitinated PTEN directly and promoted the protein degradation. Immunoblot analysis for endogenous PTEN in NEDD4 over-expressing NIH3T3 cells. Control or NEDD4 expression vector were transfected and incubated for 48 h. β-actin (ACTB) was blotted as the loading control. (C) Over-expression of NEDD4 in NIH3T3 cells enhanced the phosphorylation level of Akt. Control or NEDD4 expression vector were transfected and incubated for 48 h. Phosphor-Akt and total Akt were detected by immunoblotting. β-actin (ACTB) was blotted as the loading control.
Figure Legend Snippet: NEDD4 activated Akt signaling pathway through diminished PTEN protein level in fibroblasts. (A) In vitro ubiquitination assay of PTEN by NEDD4. NEDD4 ubiquitinated PTEN directly. (B) NEDD4 ubiquitinated PTEN directly and promoted the protein degradation. Immunoblot analysis for endogenous PTEN in NEDD4 over-expressing NIH3T3 cells. Control or NEDD4 expression vector were transfected and incubated for 48 h. β-actin (ACTB) was blotted as the loading control. (C) Over-expression of NEDD4 in NIH3T3 cells enhanced the phosphorylation level of Akt. Control or NEDD4 expression vector were transfected and incubated for 48 h. Phosphor-Akt and total Akt were detected by immunoblotting. β-actin (ACTB) was blotted as the loading control.

Techniques Used: In Vitro, Ubiquitin Assay, Expressing, Plasmid Preparation, Transfection, Incubation, Over Expression

31) Product Images from "DAZAP1 regulates the splicing of Crem, Crisp2 and Pot1a transcripts"

Article Title: DAZAP1 regulates the splicing of Crem, Crisp2 and Pot1a transcripts

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt746

EMSA assays on DAZAP1-RNA binding. ( A ) Sequences of the RNA probes from the three DAZAP1 target genes. Deleted regions are indicated with doted lines. ( B ) EMSA gel patterns. 32 P-labelled probes were incubated with buffer only (−) or 500 nM GST-DAZAP1 (+) and analysed by 5% native PAGE. Determination of the dissociation constants of the binding between DAZAP1 and the various RNA probes is shown in Supplementary Figure S4 .
Figure Legend Snippet: EMSA assays on DAZAP1-RNA binding. ( A ) Sequences of the RNA probes from the three DAZAP1 target genes. Deleted regions are indicated with doted lines. ( B ) EMSA gel patterns. 32 P-labelled probes were incubated with buffer only (−) or 500 nM GST-DAZAP1 (+) and analysed by 5% native PAGE. Determination of the dissociation constants of the binding between DAZAP1 and the various RNA probes is shown in Supplementary Figure S4 .

Techniques Used: RNA Binding Assay, Incubation, Clear Native PAGE, Binding Assay

Mapping DAZAP1 binding sites and splicing regulatory regions in the target genes. ( A ) Crem , ( B ) Crisp2 and ( C ) Pot1a . For each section dedicated to a specific gene, the sequence of the regulated exon (in Capital bold letters) and its flanking intronic regions (in low case) is shown at top. EMSA probes (P1–P5) are indicated with underlying double-headed arrows, and segments deleted in the various minigene deletion constructs (A– E ) are boxed. The left panel below the sequence shows the EMSA gel patterns. 32 P-labelled probes, indicated at top, were incubated with buffer only (mock), GST, or GST-DAZAP1 and analysed by 5% native PAGE. The middle panel shows RT-PCR gel patterns of splicing assays of the various minigene deletion constructs. The PSI of each reaction is shown below the gel lane. The right panel shows the relative change of PSI by DAZAP1, calculated from the middle panel by dividing the PSI value in the presence of exogenous DAZAP1 with that in the absence of exogenous DAZAP1. Three independent experiments were performed, and statistical significance of the differences was calculated using the paired t test. * P
Figure Legend Snippet: Mapping DAZAP1 binding sites and splicing regulatory regions in the target genes. ( A ) Crem , ( B ) Crisp2 and ( C ) Pot1a . For each section dedicated to a specific gene, the sequence of the regulated exon (in Capital bold letters) and its flanking intronic regions (in low case) is shown at top. EMSA probes (P1–P5) are indicated with underlying double-headed arrows, and segments deleted in the various minigene deletion constructs (A– E ) are boxed. The left panel below the sequence shows the EMSA gel patterns. 32 P-labelled probes, indicated at top, were incubated with buffer only (mock), GST, or GST-DAZAP1 and analysed by 5% native PAGE. The middle panel shows RT-PCR gel patterns of splicing assays of the various minigene deletion constructs. The PSI of each reaction is shown below the gel lane. The right panel shows the relative change of PSI by DAZAP1, calculated from the middle panel by dividing the PSI value in the presence of exogenous DAZAP1 with that in the absence of exogenous DAZAP1. Three independent experiments were performed, and statistical significance of the differences was calculated using the paired t test. * P

Techniques Used: Binding Assay, Sequencing, Construct, Incubation, Clear Native PAGE, Reverse Transcription Polymerase Chain Reaction

32) Product Images from "KCTD1 Suppresses Canonical Wnt Signaling Pathway by Enhancing ?-catenin Degradation"

Article Title: KCTD1 Suppresses Canonical Wnt Signaling Pathway by Enhancing ?-catenin Degradation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0094343

Identification of β-catenin binding domain in KCTD1. (A) Schematic representation of KCTD1 domains, deletion and mutant constructs used for pull-down analysis and luciferase assays. And the five silent mutations in the siRNA-target sequence of wild-type KCTD1 cDNA were shown, the resulting protein mutKCTD1 confers resistance to the siRNA and no change in the amino acid sequence compared with the wild-type. (B) The full-length and truncated proteins of GST-KCTD1 were bacterially expressed, purified and detected with Western blots using mouse monoclonal anti-GST antibodies. (C) GST pull-down experiments were performed with GST, GST fusion proteins above and His-β-catenin recombinant proteins analyzed by immunoblots with mouse monoclonal antibodies against His-tag.
Figure Legend Snippet: Identification of β-catenin binding domain in KCTD1. (A) Schematic representation of KCTD1 domains, deletion and mutant constructs used for pull-down analysis and luciferase assays. And the five silent mutations in the siRNA-target sequence of wild-type KCTD1 cDNA were shown, the resulting protein mutKCTD1 confers resistance to the siRNA and no change in the amino acid sequence compared with the wild-type. (B) The full-length and truncated proteins of GST-KCTD1 were bacterially expressed, purified and detected with Western blots using mouse monoclonal anti-GST antibodies. (C) GST pull-down experiments were performed with GST, GST fusion proteins above and His-β-catenin recombinant proteins analyzed by immunoblots with mouse monoclonal antibodies against His-tag.

Techniques Used: Binding Assay, Mutagenesis, Construct, Luciferase, Sequencing, Purification, Western Blot, Recombinant

Identification of KCTD1 binding domain in β-catenin. (A) Schematic representation of protein domain structure of β-catenin and its deletion constructs used for pull-down and luciferase assays. The mutant phosphorylation sites (the Ser45 and Ser33/37/Thr41 sites) in β-catenin mutations were shown. (B) Bacterially expressed and purified His-β-catenin fusion proteins were detected with Western blots using mouse monoclonal antibodies against His-tag. (C) His-tag pull-down experiments were performed with GST-KCTD1 and the full-length or truncated proteins of His-β-catenin analyzed by Western blots using mouse monoclonal anti-GST antibodies. GST proteins were used as negative control.
Figure Legend Snippet: Identification of KCTD1 binding domain in β-catenin. (A) Schematic representation of protein domain structure of β-catenin and its deletion constructs used for pull-down and luciferase assays. The mutant phosphorylation sites (the Ser45 and Ser33/37/Thr41 sites) in β-catenin mutations were shown. (B) Bacterially expressed and purified His-β-catenin fusion proteins were detected with Western blots using mouse monoclonal antibodies against His-tag. (C) His-tag pull-down experiments were performed with GST-KCTD1 and the full-length or truncated proteins of His-β-catenin analyzed by Western blots using mouse monoclonal anti-GST antibodies. GST proteins were used as negative control.

Techniques Used: Binding Assay, Construct, Luciferase, Mutagenesis, Purification, Western Blot, Negative Control

33) Product Images from "The Mediator subunit MED1/TRAP220 is required for optimal glucocorticoid receptor-mediated transcription activation"

Article Title: The Mediator subunit MED1/TRAP220 is required for optimal glucocorticoid receptor-mediated transcription activation

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkm661

The NR boxes of MED1/TRAP220 interact with GR-LBD in a ligand-dependent manner. ( A ) Schematic representation of GR. ( B ) Schematic representation of MED1/TRAP220 and MED1/TRAP220 mutant proteins fused with GST. Numbers indicate positions of amino acid residues, black bars represent NR (LXXLL) boxes, shaded bars represent NR mutants (LXXAA). ( C ) Comassie staining of purified GST-MED1/TRAP220 fusion proteins. ( D ) Binding of MED1/TRAP220 NR to GR domains. Glutathione-Sepharose beads containing immobilized GST or GST-MED1/TRAP220NR (amino acids 580–701) proteins were incubated with in vitro translated, 35 S-labelled GR-AF1 or GR-LBD proteins (shown in A) as indicated and bound proteins were resolved by SDS-PAGE and visualized by autoradiography.
Figure Legend Snippet: The NR boxes of MED1/TRAP220 interact with GR-LBD in a ligand-dependent manner. ( A ) Schematic representation of GR. ( B ) Schematic representation of MED1/TRAP220 and MED1/TRAP220 mutant proteins fused with GST. Numbers indicate positions of amino acid residues, black bars represent NR (LXXLL) boxes, shaded bars represent NR mutants (LXXAA). ( C ) Comassie staining of purified GST-MED1/TRAP220 fusion proteins. ( D ) Binding of MED1/TRAP220 NR to GR domains. Glutathione-Sepharose beads containing immobilized GST or GST-MED1/TRAP220NR (amino acids 580–701) proteins were incubated with in vitro translated, 35 S-labelled GR-AF1 or GR-LBD proteins (shown in A) as indicated and bound proteins were resolved by SDS-PAGE and visualized by autoradiography.

Techniques Used: Mutagenesis, Staining, Purification, Binding Assay, Incubation, In Vitro, SDS Page, Autoradiography

34) Product Images from "Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)"

Article Title: Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1)

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl1166

( A ) Interaction between hEXO1(K418A) and hMLH1/hMSH2. Lane 1: beads + hEXO1(K418A); 2: E. coli lysate + IVTT hEXO1(K418A); 3: GST-hMSH2 + vector; 4: GST-hMLH1 + vector; 5: GST-hMSH2 + hEXO1(K418A); 6: GST-hMSH2 + hEXO1; 7: GST-hMLH1 + hEXO1(K418A); 8: GST-hMLH1 + hEXO1; 9: hEXO1(K418A); 10: hEXO1. ( B ) Interaction between hEXO1(K418A) and importin α3. Lane 1: hEXO1(K418A); 2: beads + hEXO1(K418A); 3: α3/β + hEXO1(K418A); 4: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:0.5); 5: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:1); 6: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:2); 7: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:3); 8: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:5); 9: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:10); 10: hEXO1; 11: beads + hEXO1; 12: α3/β + hEXO1; 13: α3/β + hEXO1 + unlabeled hEXO1 (1:0.5); 14: α3/β + hEXO1 + unlabeled hEXO1 (1:1); 15: α3/β + hEXO1 + unlabeled hEXO1 (1:2); 16: α3/β + hEXO1 + unlabeled hEXO1 (1:3); 17: α3/β + hEXO1 + unlabeled hEXO1 (1:5); 18: α3/β + hEXO1 + unlabeled hEXO1 (1:10). ( C ) Relative intensity of bands shown in (B) as a function of unlabeled IVTT-hEXO1/labelled IVTT-hEXO1/hEXO1(K418A). Open circles: labeled hEXO1 competed with excess unlabeled hEXO1; closed circles: labeled hEXO1(K418A) competed with excess unlabeled hEXO1. Standard deviations are shown. Each data point in this figure represents the average from three independent experiments.
Figure Legend Snippet: ( A ) Interaction between hEXO1(K418A) and hMLH1/hMSH2. Lane 1: beads + hEXO1(K418A); 2: E. coli lysate + IVTT hEXO1(K418A); 3: GST-hMSH2 + vector; 4: GST-hMLH1 + vector; 5: GST-hMSH2 + hEXO1(K418A); 6: GST-hMSH2 + hEXO1; 7: GST-hMLH1 + hEXO1(K418A); 8: GST-hMLH1 + hEXO1; 9: hEXO1(K418A); 10: hEXO1. ( B ) Interaction between hEXO1(K418A) and importin α3. Lane 1: hEXO1(K418A); 2: beads + hEXO1(K418A); 3: α3/β + hEXO1(K418A); 4: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:0.5); 5: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:1); 6: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:2); 7: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:3); 8: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:5); 9: α3/β + hEXO1(K418A) + unlabeled hEXO1 (1:10); 10: hEXO1; 11: beads + hEXO1; 12: α3/β + hEXO1; 13: α3/β + hEXO1 + unlabeled hEXO1 (1:0.5); 14: α3/β + hEXO1 + unlabeled hEXO1 (1:1); 15: α3/β + hEXO1 + unlabeled hEXO1 (1:2); 16: α3/β + hEXO1 + unlabeled hEXO1 (1:3); 17: α3/β + hEXO1 + unlabeled hEXO1 (1:5); 18: α3/β + hEXO1 + unlabeled hEXO1 (1:10). ( C ) Relative intensity of bands shown in (B) as a function of unlabeled IVTT-hEXO1/labelled IVTT-hEXO1/hEXO1(K418A). Open circles: labeled hEXO1 competed with excess unlabeled hEXO1; closed circles: labeled hEXO1(K418A) competed with excess unlabeled hEXO1. Standard deviations are shown. Each data point in this figure represents the average from three independent experiments.

Techniques Used: Plasmid Preparation, Labeling

35) Product Images from "Protein kinase DYRK2 is an E3-ligase specific molecular assembler"

Article Title: Protein kinase DYRK2 is an E3-ligase specific molecular assembler

Journal: Nature cell biology

doi: 10.1038/ncb1848

DYRK2 phosphorylates Katanin (a) An in vitro kinase assay was performed with a bacterial expressed GST-Katanin and immunoprecipitated wild-type or Kinase inactive DYRK2. (b) An in vitro kinase assay was performed with a bacterial expressed GST-Katanin with immunoprecipitates prepared by using EDD, DDB1, VPRBP, DYRK2 and Cul4A antibodies. (c) The alignment of potential Katanin phosphorylation sites with DYRK2 consensus sequence is presented. Bold lettering indicates the phosphorylated residue. (d) In vitro DYRK2 kinase assays were conducted using different bacterially expressed GST-Katanin phosphorylation site mutants as indicated. (e) The in vivo phosphorylation of Katanin was detected by immunoblotting with anti-phospho-serine or anti-phospho-threonine specific antibodies following immunoprecipitation using control IgG or Katanin antibodies. (f) The in vivo phosphorylation of wild-type Katanin and the phospho Katanin mutant (AAA) was assessed by immunoblotting with phospho-serine or phospho threonine antibodies following anti-Myc immunoprecipitation of extracts prepared from 293T cells expressing Myc-tagged wild-type or mutant Katanin. IgH indicates IgG heavy chain.
Figure Legend Snippet: DYRK2 phosphorylates Katanin (a) An in vitro kinase assay was performed with a bacterial expressed GST-Katanin and immunoprecipitated wild-type or Kinase inactive DYRK2. (b) An in vitro kinase assay was performed with a bacterial expressed GST-Katanin with immunoprecipitates prepared by using EDD, DDB1, VPRBP, DYRK2 and Cul4A antibodies. (c) The alignment of potential Katanin phosphorylation sites with DYRK2 consensus sequence is presented. Bold lettering indicates the phosphorylated residue. (d) In vitro DYRK2 kinase assays were conducted using different bacterially expressed GST-Katanin phosphorylation site mutants as indicated. (e) The in vivo phosphorylation of Katanin was detected by immunoblotting with anti-phospho-serine or anti-phospho-threonine specific antibodies following immunoprecipitation using control IgG or Katanin antibodies. (f) The in vivo phosphorylation of wild-type Katanin and the phospho Katanin mutant (AAA) was assessed by immunoblotting with phospho-serine or phospho threonine antibodies following anti-Myc immunoprecipitation of extracts prepared from 293T cells expressing Myc-tagged wild-type or mutant Katanin. IgH indicates IgG heavy chain.

Techniques Used: In Vitro, Kinase Assay, Immunoprecipitation, Sequencing, In Vivo, Mutagenesis, Expressing

Katanin p60 is the ubiquitination substrate for EDVP E3 ligase complex (a) Control (IgG) or anti-FLAG immunoprecipitates were prepared from 293T cells transfected with plasmid encoding a triple tagged Katanin. Western blotting was conducted using indicated antibodies to show a specific interaction between the DYRK2-EDVP complex and Katanin p60. (b) Bacterially expressed recombinant MBP-tagged EDD, DDB1 or VPRBP bound to amylose sepharose beads were incubated with recombinant GST-Katanin and the association of Katanin was detected by western blotting with anti-GST antibody. The expression of MBP-fusion proteins was detected by anti-MBP antibody. (c) HeLa cells were transfected with either control siRNA or VPRBP siRNA and the association of EDD and DDB1 with Katanin was assessed by immunoblotting with their respective antibodies after immunoprecipitation using anti-Katanin antibody. (d) HeLa cells were transfected with different siRNAs as indicated. Cell lysates prepared after 5 hour MG132 (10µM) treatment were subjected to immunoprecipitaton using anti-Katanin antibodies. The ubiquitinated Katanin was detected with anti-ubiquitin antibody. The protein expression and the specificity of different siRNAs were confirmed by immunoblotting of cell extracts using antibodies as indicated. (e) HeLa cells transfected with EDD specific siRNA were retransfected with either siRNA resistant wild type EDD (SiR-EDD WT) or catalytically inactive EDD (SiR-EDD C/A). Ubiquitination of Katanin was assessed by immunoblotting with anti-ubiquitin antibody after immunoprecipitating with anti-Katanin antibody. The expression of endogenous EDD and the transfected siRNA resistant EDD was assessed by immunoblotting with anti-EDD antibody.
Figure Legend Snippet: Katanin p60 is the ubiquitination substrate for EDVP E3 ligase complex (a) Control (IgG) or anti-FLAG immunoprecipitates were prepared from 293T cells transfected with plasmid encoding a triple tagged Katanin. Western blotting was conducted using indicated antibodies to show a specific interaction between the DYRK2-EDVP complex and Katanin p60. (b) Bacterially expressed recombinant MBP-tagged EDD, DDB1 or VPRBP bound to amylose sepharose beads were incubated with recombinant GST-Katanin and the association of Katanin was detected by western blotting with anti-GST antibody. The expression of MBP-fusion proteins was detected by anti-MBP antibody. (c) HeLa cells were transfected with either control siRNA or VPRBP siRNA and the association of EDD and DDB1 with Katanin was assessed by immunoblotting with their respective antibodies after immunoprecipitation using anti-Katanin antibody. (d) HeLa cells were transfected with different siRNAs as indicated. Cell lysates prepared after 5 hour MG132 (10µM) treatment were subjected to immunoprecipitaton using anti-Katanin antibodies. The ubiquitinated Katanin was detected with anti-ubiquitin antibody. The protein expression and the specificity of different siRNAs were confirmed by immunoblotting of cell extracts using antibodies as indicated. (e) HeLa cells transfected with EDD specific siRNA were retransfected with either siRNA resistant wild type EDD (SiR-EDD WT) or catalytically inactive EDD (SiR-EDD C/A). Ubiquitination of Katanin was assessed by immunoblotting with anti-ubiquitin antibody after immunoprecipitating with anti-Katanin antibody. The expression of endogenous EDD and the transfected siRNA resistant EDD was assessed by immunoblotting with anti-EDD antibody.

Techniques Used: Transfection, Plasmid Preparation, Western Blot, Recombinant, Incubation, Expressing, Immunoprecipitation

DYRK2 kinase activity is required for the regulation of Katanin degradation (a) Myc-tagged wild-type or phosphomutant of Katanin was expressed in HeLa cells along with FLAG-VPRBP and HA-Ub. The levels of Katanin ubiquitination were evaluated by anti-HA immunoblotting following immunoprecipitation of Katanin from the cell extracts. (b) In vitro reconstitution experiments were performed using GST-Katanin as a substrate in the presence of recombinant ubiquitin, E1 (UBE1), E2 (UbcH5), MBP-tagged EDD, EDD C/A, DDB1, VPRBP and DYRK2 with various combinations as indicated. Ubiquitinated species of Katanin and GST-Katanin were detected by immunoblotting with anti-ubiquitin and anti-GST antibodies respectively. (c) In vitro reconstitution experiments were performed similar to figure 6B , using either wild type (WT) GST-Katanin or Katanin-AAA mutant as a substrate in the presence of various recombinant proteins as indicated. Ubiquitinated species of Katanin and GST-Katanin were detected by immunoblotting with anti-ubiquitin and anti-GST antibodies respectively. (d) The effect of DYRK2 kinase activity and Katanin phosphorylation on the regulation of Katanin protein levels was assessed by transient transfection experiments. 293T cells were transfected with the indicated expression vectors for DYRK2 and Katanin, and the protein levels were estimated by immunoblotting 24 hours post-transfection.
Figure Legend Snippet: DYRK2 kinase activity is required for the regulation of Katanin degradation (a) Myc-tagged wild-type or phosphomutant of Katanin was expressed in HeLa cells along with FLAG-VPRBP and HA-Ub. The levels of Katanin ubiquitination were evaluated by anti-HA immunoblotting following immunoprecipitation of Katanin from the cell extracts. (b) In vitro reconstitution experiments were performed using GST-Katanin as a substrate in the presence of recombinant ubiquitin, E1 (UBE1), E2 (UbcH5), MBP-tagged EDD, EDD C/A, DDB1, VPRBP and DYRK2 with various combinations as indicated. Ubiquitinated species of Katanin and GST-Katanin were detected by immunoblotting with anti-ubiquitin and anti-GST antibodies respectively. (c) In vitro reconstitution experiments were performed similar to figure 6B , using either wild type (WT) GST-Katanin or Katanin-AAA mutant as a substrate in the presence of various recombinant proteins as indicated. Ubiquitinated species of Katanin and GST-Katanin were detected by immunoblotting with anti-ubiquitin and anti-GST antibodies respectively. (d) The effect of DYRK2 kinase activity and Katanin phosphorylation on the regulation of Katanin protein levels was assessed by transient transfection experiments. 293T cells were transfected with the indicated expression vectors for DYRK2 and Katanin, and the protein levels were estimated by immunoblotting 24 hours post-transfection.

Techniques Used: Activity Assay, Immunoprecipitation, In Vitro, Recombinant, Mutagenesis, Transfection, Expressing

Identification of EDD-DDB1-VPRBP as DYRK2 associated proteins (a) Tandem affinity purification of DYRK2-containg protein complexes was conducted using 293T cells stably expressing triple tagged DYRK2. Associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. The proteins and the number of peptides identified by mass spectrometry analysis are shown in the table on the right and also in supplementary data ( Supplemental Table 1 ) (b) Immunoprecipitation using control IgG or anti-FLAG (DYRK2) antibody were performed using extracts prepared from 293T derivative cells stably expressing FLAG-tagged DYRK2. The presence of EDD, DDB1, VPRBP, Cul4A or Roc1 in these immunoprecipitates was evaluated by immunoblotting with their respective antibodies. (c) Reverse co-immunoprecipitation experiments were performed using anti-EDD, anti-Cul4A, anti-DDB1 and anti-VPRBP antibodies and the associated endogenous DYRK2 and other indicated proteins was identified by Western blotting using their respective antibodies. (d) GST pull down assay was performed using immobilized control GST or GST-DYRK2 fusion proteins on agarose beads and incubated with extracts prepared from 293T cells. The interaction of EDD, DDB1, VPRBP or Cul4A with DYRK2 was assessed by immunoblotting with their respective antibodies.
Figure Legend Snippet: Identification of EDD-DDB1-VPRBP as DYRK2 associated proteins (a) Tandem affinity purification of DYRK2-containg protein complexes was conducted using 293T cells stably expressing triple tagged DYRK2. Associated proteins were separated by SDS-PAGE and visualized by Coomassie staining. The proteins and the number of peptides identified by mass spectrometry analysis are shown in the table on the right and also in supplementary data ( Supplemental Table 1 ) (b) Immunoprecipitation using control IgG or anti-FLAG (DYRK2) antibody were performed using extracts prepared from 293T derivative cells stably expressing FLAG-tagged DYRK2. The presence of EDD, DDB1, VPRBP, Cul4A or Roc1 in these immunoprecipitates was evaluated by immunoblotting with their respective antibodies. (c) Reverse co-immunoprecipitation experiments were performed using anti-EDD, anti-Cul4A, anti-DDB1 and anti-VPRBP antibodies and the associated endogenous DYRK2 and other indicated proteins was identified by Western blotting using their respective antibodies. (d) GST pull down assay was performed using immobilized control GST or GST-DYRK2 fusion proteins on agarose beads and incubated with extracts prepared from 293T cells. The interaction of EDD, DDB1, VPRBP or Cul4A with DYRK2 was assessed by immunoblotting with their respective antibodies.

Techniques Used: Affinity Purification, Stable Transfection, Expressing, SDS Page, Staining, Mass Spectrometry, Immunoprecipitation, Western Blot, Pull Down Assay, Incubation

36) Product Images from "Hsp40 Couples with the CSP? Chaperone Complex upon Induction of the Heat Shock Response"

Article Title: Hsp40 Couples with the CSP? Chaperone Complex upon Induction of the Heat Shock Response

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004595

Western analysis showing the association of Hsp40 with CSPα 1-112 and CSPα 1-198 before and after heat shock. (A B) CAD cells were heat shocked for 30 minutes at 42°C and allowed to recover for 5 hours. GST fusion proteins of (A) CSPα 1-112 and (B) CSPα 1-198 were immobilized on glutathione-sepharose and incubated in the presence of 110 µg of control or heat shocked CAD cell homogenate in the presence or absence of 2 mM ATP or GDP. The lane indicated as (H) is 40 µg of cell homogenate loaded directly on the gel. The beads were washed and bound proteins were eluted in sample buffer, fractionated by SDS-PAGE and subjected to Western blot analysis. Hsp70, Hsc70, Hsp25, Hsp40, and Hsp90 were detected by Western analysis.
Figure Legend Snippet: Western analysis showing the association of Hsp40 with CSPα 1-112 and CSPα 1-198 before and after heat shock. (A B) CAD cells were heat shocked for 30 minutes at 42°C and allowed to recover for 5 hours. GST fusion proteins of (A) CSPα 1-112 and (B) CSPα 1-198 were immobilized on glutathione-sepharose and incubated in the presence of 110 µg of control or heat shocked CAD cell homogenate in the presence or absence of 2 mM ATP or GDP. The lane indicated as (H) is 40 µg of cell homogenate loaded directly on the gel. The beads were washed and bound proteins were eluted in sample buffer, fractionated by SDS-PAGE and subjected to Western blot analysis. Hsp70, Hsc70, Hsp25, Hsp40, and Hsp90 were detected by Western analysis.

Techniques Used: Western Blot, Incubation, SDS Page

37) Product Images from "Human CtIP Mediates Cell Cycle Control of DNA End Resection and Double Strand Break Repair *Human CtIP Mediates Cell Cycle Control of DNA End Resection and Double Strand Break Repair * S⃞"

Article Title: Human CtIP Mediates Cell Cycle Control of DNA End Resection and Double Strand Break Repair *Human CtIP Mediates Cell Cycle Control of DNA End Resection and Double Strand Break Repair * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M808906200

CtIP mutations affect DSB processing. A , cells expressing CtIP variants were treated with DMSO (-) or 25 μ m roscovitine ( Rosc. )(+) and then irradiated with 10 Gy of IR. One h later, cells were immunostained for RPA or γH2AX. Averages and standard deviations ( error bars ) of three independent experiments are shown. At least 200 cells were counted per experiment. B , representative images of cells treated in A. C , the number of RPA foci per cell in cells expressing the GFP-CtIP-T847E mutant in the presence or absence of the CDK inhibitor roscovitine. Error bars , standard deviations. D , an immunoblot of protein extracts, collected 1 h after irradiation (10 Gy), of cells expressing the indicated GFP-CtIP fusions. Panels to the left and right contain samples derived from cells treated in the absence or presence of roscovitine, respectively.
Figure Legend Snippet: CtIP mutations affect DSB processing. A , cells expressing CtIP variants were treated with DMSO (-) or 25 μ m roscovitine ( Rosc. )(+) and then irradiated with 10 Gy of IR. One h later, cells were immunostained for RPA or γH2AX. Averages and standard deviations ( error bars ) of three independent experiments are shown. At least 200 cells were counted per experiment. B , representative images of cells treated in A. C , the number of RPA foci per cell in cells expressing the GFP-CtIP-T847E mutant in the presence or absence of the CDK inhibitor roscovitine. Error bars , standard deviations. D , an immunoblot of protein extracts, collected 1 h after irradiation (10 Gy), of cells expressing the indicated GFP-CtIP fusions. Panels to the left and right contain samples derived from cells treated in the absence or presence of roscovitine, respectively.

Techniques Used: Expressing, Irradiation, Recombinase Polymerase Amplification, Mutagenesis, Derivative Assay

Functional effects of mutating Thr-847 of CtIP. A , alignment of the region conserved among Sae2/CtIP orthologues. Arrows show the position of the conserved CtIP Thr-847 and Sae2 Ser-267. A. thaliana, Arabidopsis thaliana ; C. elegans, Caenorhabditis elegans ; P. nodorum, Phaeosphaeria nodorum ; C. globosum, Chaetomium globosum ; N. crassa, Neurospora crassa ; C. neoformans, Cryptococcus neoformans ; Y. lipolytica, Yarrowia lipolytica ; A. gossypii, Ashbya gossypii. B , expression levels of GFP-CtIP derivatives in stably transfected clones before ( left ) or after ( right ) siRNA depletion of endogenous CtIP ( siCtIP ). C , representative confocal microscope images of cells expressing wild-type or T847A CtIP variants after immunostaining with a phospho-specific antibody raised against phosphorylated Thr-847. Cells were incubated in the presence of the CDK inhibitor roscovitine where indicated. D , a GST-fused wild-type or T847A mutant CtIP C-terminal fragment (residues 750-897) was affinity-purified with glutathione-Sepharose 4B and then incubated with [γ- 32 P]ATP in the presence or absence of recombinant CDK2/cyclin A, separated by 10% SDS-PAGE, and transferred to nitrocellulose membrane. Proteins were detected with an anti-GST antibody ( bottom ), and phosphorylation was visualized by autoradiography (CDK assay; top ).
Figure Legend Snippet: Functional effects of mutating Thr-847 of CtIP. A , alignment of the region conserved among Sae2/CtIP orthologues. Arrows show the position of the conserved CtIP Thr-847 and Sae2 Ser-267. A. thaliana, Arabidopsis thaliana ; C. elegans, Caenorhabditis elegans ; P. nodorum, Phaeosphaeria nodorum ; C. globosum, Chaetomium globosum ; N. crassa, Neurospora crassa ; C. neoformans, Cryptococcus neoformans ; Y. lipolytica, Yarrowia lipolytica ; A. gossypii, Ashbya gossypii. B , expression levels of GFP-CtIP derivatives in stably transfected clones before ( left ) or after ( right ) siRNA depletion of endogenous CtIP ( siCtIP ). C , representative confocal microscope images of cells expressing wild-type or T847A CtIP variants after immunostaining with a phospho-specific antibody raised against phosphorylated Thr-847. Cells were incubated in the presence of the CDK inhibitor roscovitine where indicated. D , a GST-fused wild-type or T847A mutant CtIP C-terminal fragment (residues 750-897) was affinity-purified with glutathione-Sepharose 4B and then incubated with [γ- 32 P]ATP in the presence or absence of recombinant CDK2/cyclin A, separated by 10% SDS-PAGE, and transferred to nitrocellulose membrane. Proteins were detected with an anti-GST antibody ( bottom ), and phosphorylation was visualized by autoradiography (CDK assay; top ).

Techniques Used: Functional Assay, Expressing, Stable Transfection, Transfection, Clone Assay, Microscopy, Immunostaining, Incubation, Mutagenesis, Affinity Purification, Recombinant, SDS Page, Autoradiography

38) Product Images from "Comparative functional analysis of Jembrana disease virus Tat protein on lentivirus long terminal repeat promoters: evidence for flexibility at its N-terminus"

Article Title: Comparative functional analysis of Jembrana disease virus Tat protein on lentivirus long terminal repeat promoters: evidence for flexibility at its N-terminus

Journal: Virology Journal

doi: 10.1186/1743-422X-6-179

Interaction of jTat with CycT1 in vitro and in vivo . (A) Interaction of hTat and jTat with mammalian CycT1s. GST and GST-tagged proteins were immobilized on beads and incubated with the cell lysates as described in Methods. The pull-down complexes and 5% of cell lysate input were analyzed by western-blotting using anti-Flag antibody. The coomassie blue staining shows 10% of the amounts of the purified proteins utilized in this experiment. Numbers mark the molecular weight standards (MW). (B) Schematic representation of mammalian two-hybrid constructs. jAD; jTat residues 1-67. JH; jTat 1-67 fused to hTat 48-72. (C) HeLa cells were co-transfected with 500 ng J-NFκB or H-NFκB, 500 ng of the indicated Gal4 BD plasmid and 250 ng pFR- luc . Fold-induction shows the relative activity of pFR- luc reporter and reflects binding affinity between Tat and its cofactor. (D) HIV LTR activation in 3T3 cells by indicated Tats in the absence or presence of hCycT1. The amount of transfected pCMV-Tag2B-hCycT1 was 50 ng. T1; Cyclin T1 residues 1-272.
Figure Legend Snippet: Interaction of jTat with CycT1 in vitro and in vivo . (A) Interaction of hTat and jTat with mammalian CycT1s. GST and GST-tagged proteins were immobilized on beads and incubated with the cell lysates as described in Methods. The pull-down complexes and 5% of cell lysate input were analyzed by western-blotting using anti-Flag antibody. The coomassie blue staining shows 10% of the amounts of the purified proteins utilized in this experiment. Numbers mark the molecular weight standards (MW). (B) Schematic representation of mammalian two-hybrid constructs. jAD; jTat residues 1-67. JH; jTat 1-67 fused to hTat 48-72. (C) HeLa cells were co-transfected with 500 ng J-NFκB or H-NFκB, 500 ng of the indicated Gal4 BD plasmid and 250 ng pFR- luc . Fold-induction shows the relative activity of pFR- luc reporter and reflects binding affinity between Tat and its cofactor. (D) HIV LTR activation in 3T3 cells by indicated Tats in the absence or presence of hCycT1. The amount of transfected pCMV-Tag2B-hCycT1 was 50 ng. T1; Cyclin T1 residues 1-272.

Techniques Used: In Vitro, In Vivo, Incubation, Western Blot, Staining, Purification, Molecular Weight, Construct, Transfection, Plasmid Preparation, Activity Assay, Binding Assay, Activation Assay

39) Product Images from "CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex"

Article Title: CLASP1 and CLASP2 bind to EB1 and regulate microtubule plus-end dynamics at the cell cortex

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200405094

The COOH-terminal domain of CLASP2 is responsible for association with the cell cortex and Golgi complex. (A–F) TIRF microscopy images of live HeLa cells, expressing GFP-CLASP1α (A), GFP-CLASP2γ (B), GFP-α-tubulin (C and D), or EB3-GFP (E and F). Cells were either not treated with siRNAs (A and B), or treated for 72 h with control (C and E) or CLASP1+2#B siRNAs (D and F). The contrast is inverted. Bars, 10 μm. (G) Schematic representation of CLASP2γ and the relevant deletion mutants. (H) HeLa cells were transfected with GFP-CLASP2-C and stained for the Golgi marker GM130. Bar, 10 μm. (I) HeLa cells were transfected with GFP-CLASP2 or GFP-CLASP2-ΔC and were either fixed directly or treated with 10 μM nocodazole for 1 h before fixation and stained for α-tubulin. Bars, 10 μm.
Figure Legend Snippet: The COOH-terminal domain of CLASP2 is responsible for association with the cell cortex and Golgi complex. (A–F) TIRF microscopy images of live HeLa cells, expressing GFP-CLASP1α (A), GFP-CLASP2γ (B), GFP-α-tubulin (C and D), or EB3-GFP (E and F). Cells were either not treated with siRNAs (A and B), or treated for 72 h with control (C and E) or CLASP1+2#B siRNAs (D and F). The contrast is inverted. Bars, 10 μm. (G) Schematic representation of CLASP2γ and the relevant deletion mutants. (H) HeLa cells were transfected with GFP-CLASP2-C and stained for the Golgi marker GM130. Bar, 10 μm. (I) HeLa cells were transfected with GFP-CLASP2 or GFP-CLASP2-ΔC and were either fixed directly or treated with 10 μM nocodazole for 1 h before fixation and stained for α-tubulin. Bars, 10 μm.

Techniques Used: Microscopy, Expressing, Transfection, Staining, Marker

40) Product Images from "Effects of Structure of Rho GTPase-activating Protein DLC-1 on Cell Morphology and Migration *Effects of Structure of Rho GTPase-activating Protein DLC-1 on Cell Morphology and Migration * S⃞"

Article Title: Effects of Structure of Rho GTPase-activating Protein DLC-1 on Cell Morphology and Migration *Effects of Structure of Rho GTPase-activating Protein DLC-1 on Cell Morphology and Migration * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M800617200

SAM domain deleted DLC-1 shows enhanced catalytic activity for RhoA. A , bacterially expressed full-length, SAM domain deleted (amino acids 77-1091), and RhoGAP domain fragment (amino acids 609-878) of DLC-1 were purified for analysis of in vitro GAP activity. Purified GST-RhoA fusion proteins were preloaded with GTP, and GTP hydrolysis was monitored by incubation with a phosphate-binding protein that undergoes a major increase in fluorescence upon binding inorganic phosphate. B , GTP hydrolysis activities of DLC-1 constructs. C , GTP loading of RhoA in cells was monitored by a Rhotekin pull-down assay as described under “Experimental Procedures.”
Figure Legend Snippet: SAM domain deleted DLC-1 shows enhanced catalytic activity for RhoA. A , bacterially expressed full-length, SAM domain deleted (amino acids 77-1091), and RhoGAP domain fragment (amino acids 609-878) of DLC-1 were purified for analysis of in vitro GAP activity. Purified GST-RhoA fusion proteins were preloaded with GTP, and GTP hydrolysis was monitored by incubation with a phosphate-binding protein that undergoes a major increase in fluorescence upon binding inorganic phosphate. B , GTP hydrolysis activities of DLC-1 constructs. C , GTP loading of RhoA in cells was monitored by a Rhotekin pull-down assay as described under “Experimental Procedures.”

Techniques Used: Activity Assay, Purification, In Vitro, Incubation, Binding Assay, Fluorescence, Construct, Pull Down Assay

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Filtration:

Article Title: Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4
Article Snippet: .. The column was washed with 1% Triton X-100 in PBS followed by PBS alone, after which thrombin (Sigma-Aldrich, St. Louis, MI, USA) was loaded onto the column and incubated at room temperature overnight to cleave rCan f 6 and rFel d 4 from GST. rCan f 6 and rFel d 4 were eluted by loading PBS onto the column and then subjected to further purification by gel filtration chromatography using HiLoad 16/600 Superdex 75 pg (GE Healthcare) with PBS. ..

Chromatography:

Article Title: Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4
Article Snippet: .. The column was washed with 1% Triton X-100 in PBS followed by PBS alone, after which thrombin (Sigma-Aldrich, St. Louis, MI, USA) was loaded onto the column and incubated at room temperature overnight to cleave rCan f 6 and rFel d 4 from GST. rCan f 6 and rFel d 4 were eluted by loading PBS onto the column and then subjected to further purification by gel filtration chromatography using HiLoad 16/600 Superdex 75 pg (GE Healthcare) with PBS. ..

Autoradiography:

Article Title: Ehrlichia type IV secretion system effector Etf-2 binds to active RAB5 and delays endosome maturation
Article Snippet: .. Samples were taken at the indicated time and were subjected to TLC to separate the hydrolysis product [α-32 P]GDP from the substrate [α-32 P]GTP, followed by autoradiography and quantification using a PhosphorImager (GE Healthcare Life Sciences). ..

Purification:

Article Title: Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4
Article Snippet: .. The column was washed with 1% Triton X-100 in PBS followed by PBS alone, after which thrombin (Sigma-Aldrich, St. Louis, MI, USA) was loaded onto the column and incubated at room temperature overnight to cleave rCan f 6 and rFel d 4 from GST. rCan f 6 and rFel d 4 were eluted by loading PBS onto the column and then subjected to further purification by gel filtration chromatography using HiLoad 16/600 Superdex 75 pg (GE Healthcare) with PBS. ..

Article Title: The LIM protein Ajuba recruits DBC1 and CBP/p300 to acetylate ERα and enhances ERα target gene expression in breast cancer cells
Article Snippet: .. GST-tagged ERα or DBC1and His tagged Ajuba were expressed in BL21 respectively, and purified by Glutathione Sepharose beads (17-0756-01, GE Healthcare) or Ni-beads (17-5318-06, GE Healthcare). .. For the in vitro binding assays, the purified proteins of GST-ERα or DBC1 were mixed with His-Ajuba and E2 (100nM) was added into the mixture for 12 hours.

Article Title: STK25-induced inhibition of aerobic glycolysis via GOLPH3-mTOR pathway suppresses cell proliferation in colorectal cancer
Article Snippet: .. His-STK25, GST-GOLPH3, and GST were expressed and purified in accordance with the manufacturer’s instructions (Amersham). .. Then, 10 μg of His-STK25 was mixed with 10 μg of GST-GOLPH3 or GST and incubated with glutathione sepharose 4B beads (GE Healthcare) and Ni-NTA agarose (QIAGEN), respectively, for the GST pull-down and His-tag pull-down assays.

Article Title: BCAS2 Enhances Carcinogenic Effects of Estrogen Receptor Alpha in Breast Cancer Cells
Article Snippet: .. Glutathione-S-Transferase (GST) Pull-Down Assays GST fusion proteins: pGST-ERα Full (aa 1-595), pGST-ERα N (aa 1-180), pGST-ERα LBD (aa 264-595) were expressed in E. coli strain BL21, induced with 0.2% l -arabinose and purified with glutathione-Sepharose beads, according to manufacturer’s instructions (GE Healthcare). .. Biotin labeling and in vitro translation of BCAS2 was performed using the TNT® T7 Quick Coupled Transcription/Translation System (Promega, Madison, WI, USA).

Article Title: A phosphorylated transcription factor regulates sterol biosynthesis in Fusarium graminearum
Article Snippet: .. The GST fusion proteins encoding the FgHog1 and FgPbs2EE were expressed in E. coli DH5a and purified using glutathione-sepharose beads (GE Healthcare). .. To test in vitro binding between His- and GST-tagged proteins, 3 μg of GST-tagged protein or GST (negative control) that was still bound to the glutathione beads was mixed with 10 μg of His-tagged protein and rocked for 2 h at 4 °C.

Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation
Article Snippet: .. Then the fusion protein GST-α-syn was purified on glutathione-sepharose 4B according to the manufacturer’s instructions (GE Healthcare, Boston, MA). .. The purified GST-fusion proteins were desalted on Vivaspin 6 column from GE Healthcare, followed by dialysis into binding buffer (PBS, 1 mM MgCI2 , pH 7.4) to remove the free glutathione.

Incubation:

Article Title: Crystal structure of the dog allergen Can f 6 and structure-based implications of its cross-reactivity with the cat allergen Fel d 4
Article Snippet: .. The column was washed with 1% Triton X-100 in PBS followed by PBS alone, after which thrombin (Sigma-Aldrich, St. Louis, MI, USA) was loaded onto the column and incubated at room temperature overnight to cleave rCan f 6 and rFel d 4 from GST. rCan f 6 and rFel d 4 were eluted by loading PBS onto the column and then subjected to further purification by gel filtration chromatography using HiLoad 16/600 Superdex 75 pg (GE Healthcare) with PBS. ..

Thin Layer Chromatography:

Article Title: Ehrlichia type IV secretion system effector Etf-2 binds to active RAB5 and delays endosome maturation
Article Snippet: .. Samples were taken at the indicated time and were subjected to TLC to separate the hydrolysis product [α-32 P]GDP from the substrate [α-32 P]GTP, followed by autoradiography and quantification using a PhosphorImager (GE Healthcare Life Sciences). ..

Affinity Purification:

Article Title: PfMSA180 is a novel Plasmodium falciparum vaccine antigen that interacts with human erythrocyte integrin associated protein (CD47)
Article Snippet: .. The truncates (Tr) Tr1, residues E22 -S263 ; Tr2, A264 -D501 ; Tr3, I508 -P723 ; Tr4, A805 -P1093 ; and Tr5, L1193 -P1455 were affinity purified using a glutathione-Sepharose 4B column (GE Healthcare, Camarillo, CA). .. The captured proteins were eluted by either on-column cleavage with AcTEV protease (Thermo Fisher Scientific, Waltham, MA) (Tr1, 2 and 4), targeting a tobacco etch virus (TEV) protease recognition site located between the GST tag and the recombinant protein, or with 20 mM glutathione (Tr3 and 5).

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    GE Healthcare glutathione sepharose 4b
    α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on <t>glutathione-sepharose</t> beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.
    Glutathione Sepharose 4b, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 2034 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare glutathione sepharose 4b beads
    Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to <t>glutathione-Sepharose</t> 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.
    Glutathione Sepharose 4b Beads, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GE Healthcare glutathione sepharose resin
    The OAR of Rtf1 interacts directly with the CTR of Spt5. (A) Recombinant GST (pGEX-3X), GST-Rtf1-His 6 (pAP21), GST-Rtf1ΔOAR-His 6 (pMM26), and GST-OAR (pMM25) proteins, bound to <t>glutathione-Sepharose</t> beads, were incubated with the same amount of
    Glutathione Sepharose Resin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 198 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: Novel DNA Aptamers for Parkinson’s Disease Treatment Inhibit α-Synuclein Aggregation and Facilitate its Degradation

    doi: 10.1016/j.omtn.2018.02.011

    Figure Lengend Snippet: α-syn Aptamers Were Selected through SELEX (A) Schematic illustration of the method used for α-syn aptamer selection. GST-tagged α-syn was immobilized on glutathione-sepharose beads. The ssDNA library was incubated with the target beads for binding. Unbound oligonucleotides were washed away, and the bound ones were released by heating at 95°C. The selected binders were amplified by PCR with biotinylated primers. ssDNAs were subsequently purified from the PCR product using streptavidin-coated magnetic beads, resulting in an enriched DNA pool, which was used in the next SELEX round. After the last round, the selected ssDNAs were sequenced by deep sequencing. (B) The aptamer candidates. After deep sequencing, the two sequences with most frequently appearing were selected as the aptamer candidates. (C) Aptamer binding specificity assay by dot blotting. Five microgram samples (α-syn, GST, Aβ 42 , BSA, and three domains of α-syn) were respectively immobilized onto the nitrocellulose membrane for binding of each aptamer.

    Article Snippet: Then the fusion protein GST-α-syn was purified on glutathione-sepharose 4B according to the manufacturer’s instructions (GE Healthcare, Boston, MA).

    Techniques: Selection, Incubation, Binding Assay, Amplification, Polymerase Chain Reaction, Purification, Magnetic Beads, Sequencing

    Effect of C-terminal truncation of LKB1 on AMPK activation in cell-free assays and ACC phosphorylation and cell cycle progress in G361 melanoma cells. A , plasmids encoding GST fusions of wild type LKB1 L and a C-terminal truncation (1–343) were co-expressed with FLAG-STRADα and myc -MO25α in HEK-293 cells and purified on glutathione-Sepharose. The purified products were analyzed by Western blotting using anti-GST, anti-FLAG, and anti- myc antibodies. B , a bacterially expressed GST fusion of the AMPK-α1 kinase domain was incubated with MgATP and various concentrations of GST-LKB1·FLAG-STRADα· myc -MO25α complex purified as in A , and AMPK activity was determined after 15 min. C , phosphorylation of the AMPK target, ACC, total ACC, and expression of GFP-LKB1 assessed using an anti-GFP antibody, in G361 cells co-expressing STRADα and MO25α with free GFP (control) or GFP fusions of wild type LKB1L and a C-terminally truncated mutant (1–343). D , cell cycle analysis of GFP-expressing cells treated as in Fig. 5 C , 18 h after nocodazole treatment.

    Journal: The Journal of Biological Chemistry

    Article Title: C-terminal Phosphorylation of LKB1 Is Not Required for Regulation of AMP-activated Protein Kinase, BRSK1, BRSK2, or Cell Cycle Arrest *

    doi: 10.1074/jbc.M806152200

    Figure Lengend Snippet: Effect of C-terminal truncation of LKB1 on AMPK activation in cell-free assays and ACC phosphorylation and cell cycle progress in G361 melanoma cells. A , plasmids encoding GST fusions of wild type LKB1 L and a C-terminal truncation (1–343) were co-expressed with FLAG-STRADα and myc -MO25α in HEK-293 cells and purified on glutathione-Sepharose. The purified products were analyzed by Western blotting using anti-GST, anti-FLAG, and anti- myc antibodies. B , a bacterially expressed GST fusion of the AMPK-α1 kinase domain was incubated with MgATP and various concentrations of GST-LKB1·FLAG-STRADα· myc -MO25α complex purified as in A , and AMPK activity was determined after 15 min. C , phosphorylation of the AMPK target, ACC, total ACC, and expression of GFP-LKB1 assessed using an anti-GFP antibody, in G361 cells co-expressing STRADα and MO25α with free GFP (control) or GFP fusions of wild type LKB1L and a C-terminally truncated mutant (1–343). D , cell cycle analysis of GFP-expressing cells treated as in Fig. 5 C , 18 h after nocodazole treatment.

    Article Snippet: After purification on glutathione-Sepharose, we obtained equal yields of full-length and truncated LKB1L , and both co-purified with FLAG-STRADα and myc -MO25α as expected ( ).

    Techniques: Activation Assay, Purification, Western Blot, Incubation, Activity Assay, Expressing, Mutagenesis, Cell Cycle Assay

    Phosphorylation and activation of AMPK, BRSK1, and BRSK2 by LKB1 variants in cell-free assays. A , purification of LKB1·STRADα·MO25α complexes from HEK-293 cells. Plasmids encoding FLAG-tagged STRADα and myc -tagged MO25α were co-expressed in HEK-293 cells with the indicated variants of GST-tagged LKB1. GST fusions were purified on glutathione-Sepharose, and the products were analyzed by Western blotting using anti-GST, anti-FLAG, or anti- myc antibodies. B –E, bacterially expressed GST fusions with the kinase domains of AMPK-α1 ( B and C ), BRSK1 ( D ), or BRSK2 ( E ) were incubated with MgATP and LKB1·STRADα·MO25α complexes (50 μg·ml –1 ) purified as in A . After 15 min the incubations were analyzed for activity of AMPK ( B ), BRSK1 ( D ), or BRSK2 ( E ) and for phosphorylation of the threonine residue equivalent to Thr-172 using anti-pT172 antibody ( C –E). WT , wild type.

    Journal: The Journal of Biological Chemistry

    Article Title: C-terminal Phosphorylation of LKB1 Is Not Required for Regulation of AMP-activated Protein Kinase, BRSK1, BRSK2, or Cell Cycle Arrest *

    doi: 10.1074/jbc.M806152200

    Figure Lengend Snippet: Phosphorylation and activation of AMPK, BRSK1, and BRSK2 by LKB1 variants in cell-free assays. A , purification of LKB1·STRADα·MO25α complexes from HEK-293 cells. Plasmids encoding FLAG-tagged STRADα and myc -tagged MO25α were co-expressed in HEK-293 cells with the indicated variants of GST-tagged LKB1. GST fusions were purified on glutathione-Sepharose, and the products were analyzed by Western blotting using anti-GST, anti-FLAG, or anti- myc antibodies. B –E, bacterially expressed GST fusions with the kinase domains of AMPK-α1 ( B and C ), BRSK1 ( D ), or BRSK2 ( E ) were incubated with MgATP and LKB1·STRADα·MO25α complexes (50 μg·ml –1 ) purified as in A . After 15 min the incubations were analyzed for activity of AMPK ( B ), BRSK1 ( D ), or BRSK2 ( E ) and for phosphorylation of the threonine residue equivalent to Thr-172 using anti-pT172 antibody ( C –E). WT , wild type.

    Article Snippet: After purification on glutathione-Sepharose, we obtained equal yields of full-length and truncated LKB1L , and both co-purified with FLAG-STRADα and myc -MO25α as expected ( ).

    Techniques: Activation Assay, Purification, Western Blot, Incubation, Activity Assay

    Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

    Journal: International Journal of Molecular Sciences

    Article Title: Three Basic Residues of Intracellular Loop 3 of the Beta-1 Adrenergic Receptor Are Required for Golgin-160-Dependent Trafficking

    doi: 10.3390/ijms15022929

    Figure Lengend Snippet: Beta-1 adrenergic receptor (β1AR) binds directly to golgin-160 (1–393) . Representative gels for the purification of golgin-160 (1–393) and its binding to β1AR are shown. ( A ) The NEB IMPACT system was used to create a purified, untagged golgin-160 (1–393) following cleavage of the intein tag. DTT-induced cleavage caused enrichment of an approximately 60 kDa protein, which was specifically eluted off of the chitin column. This protein band could be detected using immunoblotting with an antibody to the N-terminus of golgin-160. Input, protein added to the chitin column; Cleaved, protein on the chitin column after addition of DTT but before elution; Eluate, protein released from the column after cleavage; *, golgin-160 (1–393) ; **, GST fusion proteins; ( B ) The purified, untagged golgin-160 head domain was incubated with purified GST or GST-β1AR L3 pre-bound to glutathione-Sepharose 4B beads. The beads were washed and bound golgin-160 (1–393) was detected by Coomassie blue staining after SDS-PAGE. Note that the samples in panel A were run on a 4%–12% gradient gel, whereas those in B were run on a 10% gel.

    Article Snippet: The soluble fraction of the lysed cells was incubated 2 h at 4 °C with 10 μg GST alone or GST-tagged golgin-160(1–393) that had been pre-conjugated to glutathione-Sepharose 4B beads.

    Techniques: Purification, Binding Assay, Incubation, Staining, SDS Page

    The OAR of Rtf1 interacts directly with the CTR of Spt5. (A) Recombinant GST (pGEX-3X), GST-Rtf1-His 6 (pAP21), GST-Rtf1ΔOAR-His 6 (pMM26), and GST-OAR (pMM25) proteins, bound to glutathione-Sepharose beads, were incubated with the same amount of

    Journal: Molecular and Cellular Biology

    Article Title: The Recruitment of the Saccharomyces cerevisiae Paf1 Complex to Active Genes Requires a Domain of Rtf1 That Directly Interacts with the Spt4-Spt5 Complex

    doi: 10.1128/MCB.00270-13

    Figure Lengend Snippet: The OAR of Rtf1 interacts directly with the CTR of Spt5. (A) Recombinant GST (pGEX-3X), GST-Rtf1-His 6 (pAP21), GST-Rtf1ΔOAR-His 6 (pMM26), and GST-OAR (pMM25) proteins, bound to glutathione-Sepharose beads, were incubated with the same amount of

    Article Snippet: Clarified lysates were incubated with 1 ml of bovine serum albumin (BSA)-blocked 50% glutathione-Sepharose resin (GE Healthcare) for 1 h at 4°C to purify GST and GST-OAR.

    Techniques: Recombinant, Incubation