Structured Review

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Mapping regions of ABIN-2 which interact with p105 and TPL-2. (A) Schematic diagram of recombinant GST-ABIN-2 fusion proteins. The positions of the ABIN homology domain (AHD) and the binding regions for TPL-2, p105, and A20 are shown. The N and C termini of the wild-type (WT) GST-ABIN-2 protein (amino acids 1 to 420) are indicated. (B and C) 293 cells were transfected with vectors encoding Myc-p105, Myc-TPL-2, or Myc-A20. Cell lysates were prepared using 1% Brij 58 <t>buffer</t> A and incubated with the indicated GST-ABIN-2 fusion proteins or GST (control) coupled to glutathione-Sepharose 4B. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.
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1) Product Images from "ABIN-2 Forms a Ternary Complex with TPL-2 and NF-?B1 p105 and Is Essential for TPL-2 Protein Stability †"

Article Title: ABIN-2 Forms a Ternary Complex with TPL-2 and NF-?B1 p105 and Is Essential for TPL-2 Protein Stability †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.24.12.5235-5248.2004

Mapping regions of ABIN-2 which interact with p105 and TPL-2. (A) Schematic diagram of recombinant GST-ABIN-2 fusion proteins. The positions of the ABIN homology domain (AHD) and the binding regions for TPL-2, p105, and A20 are shown. The N and C termini of the wild-type (WT) GST-ABIN-2 protein (amino acids 1 to 420) are indicated. (B and C) 293 cells were transfected with vectors encoding Myc-p105, Myc-TPL-2, or Myc-A20. Cell lysates were prepared using 1% Brij 58 buffer A and incubated with the indicated GST-ABIN-2 fusion proteins or GST (control) coupled to glutathione-Sepharose 4B. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.
Figure Legend Snippet: Mapping regions of ABIN-2 which interact with p105 and TPL-2. (A) Schematic diagram of recombinant GST-ABIN-2 fusion proteins. The positions of the ABIN homology domain (AHD) and the binding regions for TPL-2, p105, and A20 are shown. The N and C termini of the wild-type (WT) GST-ABIN-2 protein (amino acids 1 to 420) are indicated. (B and C) 293 cells were transfected with vectors encoding Myc-p105, Myc-TPL-2, or Myc-A20. Cell lysates were prepared using 1% Brij 58 buffer A and incubated with the indicated GST-ABIN-2 fusion proteins or GST (control) coupled to glutathione-Sepharose 4B. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.

Techniques Used: Recombinant, Binding Assay, Transfection, Incubation, Affinity Purification, SDS Page, Western Blot

Mapping interacting regions for ABIN-2 on p105 and TPL-2. (A) Schematic diagram of HA-p105 mutants. The relative positions of the Rel homology domain (RHD), ankyrin repeats (ANK), death domain (DD), and PEST region are shown. The N and C termini of the wild-type (WT) HA-p105 protein (amino acids 1 to 968) are indicated. (B) 293 cells were transfected with vectors encoding wild-type (WT) and mutant forms of HA-p105. Cell lysates, prepared using 1% Brij 58 buffer A, were incubated with GST-ABIN-2 1-429  fusion protein or GST (control) coupled to glutathione-Sepharose beads. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti. (C) GST-p105 497-968  fusion protein and GST (control) were coupled to glutathione-Sepharose beads and used to affinity purify ABIN-2-FL translated and labeled with [ 35 S]methionine in vitro. Isolated protein was detected by autoradiography of SDS-8% acrylamide gels. (D) 293 cells were transfected with vectors encoding wild-type and mutant forms of Myc-TPL-2. GST-ABIN-2 1-429  was used as an affinity ligand to isolate protein from cell lysates prepared with 1% NP-40 buffer A. Isolated protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. (E) TPL-2 398-467  peptide coupled to streptavidin-agarose beads was used as an affinity ligand to isolate ABIN-2-FL from lysates of transfected 293 cells. Bound protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting.
Figure Legend Snippet: Mapping interacting regions for ABIN-2 on p105 and TPL-2. (A) Schematic diagram of HA-p105 mutants. The relative positions of the Rel homology domain (RHD), ankyrin repeats (ANK), death domain (DD), and PEST region are shown. The N and C termini of the wild-type (WT) HA-p105 protein (amino acids 1 to 968) are indicated. (B) 293 cells were transfected with vectors encoding wild-type (WT) and mutant forms of HA-p105. Cell lysates, prepared using 1% Brij 58 buffer A, were incubated with GST-ABIN-2 1-429 fusion protein or GST (control) coupled to glutathione-Sepharose beads. Affinity-purified protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti. (C) GST-p105 497-968 fusion protein and GST (control) were coupled to glutathione-Sepharose beads and used to affinity purify ABIN-2-FL translated and labeled with [ 35 S]methionine in vitro. Isolated protein was detected by autoradiography of SDS-8% acrylamide gels. (D) 293 cells were transfected with vectors encoding wild-type and mutant forms of Myc-TPL-2. GST-ABIN-2 1-429 was used as an affinity ligand to isolate protein from cell lysates prepared with 1% NP-40 buffer A. Isolated protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting. (E) TPL-2 398-467 peptide coupled to streptavidin-agarose beads was used as an affinity ligand to isolate ABIN-2-FL from lysates of transfected 293 cells. Bound protein was resolved by SDS-PAGE (10% acrylamide) and Western blotting.

Techniques Used: Transfection, Mutagenesis, Incubation, Affinity Purification, SDS Page, Western Blot, Labeling, In Vitro, Isolation, Autoradiography

ABIN-2 preferentially interacts with a p105/TPL-2 complex. (A and B) Duplicate cultures of 293 cells were cotransfected with vectors encoding ABIN2-FL and HA-p105 or Myc-TPL-2 or with EV. Cell lysates were prepared from each duplicate culture set using either buffer A (1% NP-40) or RIPA buffer, as indicated. Lysates were resolved by SDS-PAGE (10% acrylamide) and Western blotting (top blots). HA-p105 and Myc-TPL-2 mRNA levels in total RNA were assayed by semiquantitative RT-PCR (bottom blots). The 18S rRNA amplicon was used as an internal control. (C) 293 cells were cotransfected with vectors encoding HA-p105 and TPL-2 individually or together. Transfected proteins were affinity purified from cell lysates, prepared in 1% NP-40 buffer A, using GST-ABIN-2 1-429 fusion protein coupled to glutathione-Sepharose. Isolated proteins were resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.
Figure Legend Snippet: ABIN-2 preferentially interacts with a p105/TPL-2 complex. (A and B) Duplicate cultures of 293 cells were cotransfected with vectors encoding ABIN2-FL and HA-p105 or Myc-TPL-2 or with EV. Cell lysates were prepared from each duplicate culture set using either buffer A (1% NP-40) or RIPA buffer, as indicated. Lysates were resolved by SDS-PAGE (10% acrylamide) and Western blotting (top blots). HA-p105 and Myc-TPL-2 mRNA levels in total RNA were assayed by semiquantitative RT-PCR (bottom blots). The 18S rRNA amplicon was used as an internal control. (C) 293 cells were cotransfected with vectors encoding HA-p105 and TPL-2 individually or together. Transfected proteins were affinity purified from cell lysates, prepared in 1% NP-40 buffer A, using GST-ABIN-2 1-429 fusion protein coupled to glutathione-Sepharose. Isolated proteins were resolved by SDS-PAGE (10% acrylamide) and Western blotting. α, anti.

Techniques Used: SDS Page, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification, Transfection, Affinity Purification, Isolation

2) Product Images from "Affinity pulldown of γ-secretase and associated proteins from human and rat brain"

Article Title: Affinity pulldown of γ-secretase and associated proteins from human and rat brain

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2009.00907.x

γ-secretase complex components were specifically captured by GCB pulldown from rat brain and eluted by reducing conditions. (A) Solubilized γ-secretase prepared from rat brain material was incubated with increasing concentrations of GCB and isolated with SA beads. The captured γ-secretase complex was eluted by SDS sample buffer and subject to Western blotting for the indicated γ-secretase subunit. (B) The recovery of nicastrin, PS1-NTF and PS2-CTF was estimated by quantifying the density of the respective bands and the input (10% of the total sample) by a CCD camera and appropriate software. (C) Solubilized γ-secretase prepared from rat brain material was incubated with 200 nM GCB in the presence (+) or the absence (−) of 10 μM L-685,458 and isolated with SA beads. The captured γ-secretase complex was eluted with SDS sample buffer or 100 mM DTT supplement with 0.5% CHAPSO. Eluted samples were separated by SDS-PAGE and transferred to PVDF membrane. Transferred membrane was analysed by colloidal gold staining. Elution using reducing reagent (DTT) clearly reduced non-specific binding compared to elution using SDS sample buffer. Also the elution of SA (13 kD) was reduced. (D) The same samples as in (C) were subjected to Western blotting for the indicated γ-secretase subunit. (E) Solubilized γ-secretase prepared from rat brain material was incubated with 200 nM GCB in the presence (+) or the absence (−) of 10 μM L-685,458 and isolated with SA beads. The resin was washed indicated times with buffer A with 0.5% CHAPSO at room temperature and eluted with SDS sample buffer. Eluted samples were subjected to Western blotting for the indicated γ-secretase subunit and analysed by colloidal gold staining.
Figure Legend Snippet: γ-secretase complex components were specifically captured by GCB pulldown from rat brain and eluted by reducing conditions. (A) Solubilized γ-secretase prepared from rat brain material was incubated with increasing concentrations of GCB and isolated with SA beads. The captured γ-secretase complex was eluted by SDS sample buffer and subject to Western blotting for the indicated γ-secretase subunit. (B) The recovery of nicastrin, PS1-NTF and PS2-CTF was estimated by quantifying the density of the respective bands and the input (10% of the total sample) by a CCD camera and appropriate software. (C) Solubilized γ-secretase prepared from rat brain material was incubated with 200 nM GCB in the presence (+) or the absence (−) of 10 μM L-685,458 and isolated with SA beads. The captured γ-secretase complex was eluted with SDS sample buffer or 100 mM DTT supplement with 0.5% CHAPSO. Eluted samples were separated by SDS-PAGE and transferred to PVDF membrane. Transferred membrane was analysed by colloidal gold staining. Elution using reducing reagent (DTT) clearly reduced non-specific binding compared to elution using SDS sample buffer. Also the elution of SA (13 kD) was reduced. (D) The same samples as in (C) were subjected to Western blotting for the indicated γ-secretase subunit. (E) Solubilized γ-secretase prepared from rat brain material was incubated with 200 nM GCB in the presence (+) or the absence (−) of 10 μM L-685,458 and isolated with SA beads. The resin was washed indicated times with buffer A with 0.5% CHAPSO at room temperature and eluted with SDS sample buffer. Eluted samples were subjected to Western blotting for the indicated γ-secretase subunit and analysed by colloidal gold staining.

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

3) Product Images from "The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding"

Article Title: The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkr049

( a ) Predicted domains in the native MobM protein. The three conserved motifs located in the N-terminal moiety are indicated: (i) HxxR (unknown function), (ii) NYEL (proposed catalytic region) and (iii) HxDExxPHuH (metal ion coordination). The position of the putative Leu zipper in the C-terminal moiety is also indicated. ( b ) Stages in the purification of MobMN199. Fractions of the different purification steps were analysed by electrophoresis on 15% SDS–Tris–glycine–PAA gels. Samples loaded were: uninduced cultures (lane 1); cultures induced with IPTG and rifampicin (lane 2); supernatant of a total cell lysate (lane 3); supernatant after PEI precipitation (lane 4); supernatant of the ammonium sulphate precipitation step before (lane 5) and after (lane 6) dialysis against buffer A. The sample was loaded onto a heparin–agarose column and the proteins retained were eluted by a salt gradient (covered by lane 7). Fractions containing the peak of MobMN199 were pooled, dialysed against buffer A and concentrated. M indicates the molecular weight standards (in kDa). ( c ) Purified MobMN199 was injected onto a gel filtration column, and its elution profile was recorded; the inset shows the SDS–PAGE gel with the purified protein and the molecular size markers. ( d ) Relaxation assays with wild-type MobM and the MobMN199 protein. Supercoiled pMV158 DNA samples (300 ng; 8 nM) were incubated with and without (−) full-length MobM (left) or with the short MobMN199 fragment (right) in the presence of 15 mM MnCl 2 at 30°C, 20 min. Protein concentrations used were 120, 240 and 480 nM. Generation of relaxed forms (FII) from supercoiled DNA forms (FI) was analysed by electrophoresis on 1% agarose gels without prior staining with EtBr, conditions in which forms FI′ are not resolved. The amounts of relaxed DNA forms generated by treatment of MobM and by MobMN199 were calculated by subtracting the amount of already nicked molecules (faint FII band in the untreated samples generated by mechanical shearing) from the FII-forms generated by protein treatment. The values of the protein-relaxed molecules were 25, 42 and 62%, and 28, 40 and 63% for samples treated with MobM and MobMN199, respectively. The weak band above relaxed forms FII has been observed before ( 20 ) and might correspond to relaxed DNA dimers.
Figure Legend Snippet: ( a ) Predicted domains in the native MobM protein. The three conserved motifs located in the N-terminal moiety are indicated: (i) HxxR (unknown function), (ii) NYEL (proposed catalytic region) and (iii) HxDExxPHuH (metal ion coordination). The position of the putative Leu zipper in the C-terminal moiety is also indicated. ( b ) Stages in the purification of MobMN199. Fractions of the different purification steps were analysed by electrophoresis on 15% SDS–Tris–glycine–PAA gels. Samples loaded were: uninduced cultures (lane 1); cultures induced with IPTG and rifampicin (lane 2); supernatant of a total cell lysate (lane 3); supernatant after PEI precipitation (lane 4); supernatant of the ammonium sulphate precipitation step before (lane 5) and after (lane 6) dialysis against buffer A. The sample was loaded onto a heparin–agarose column and the proteins retained were eluted by a salt gradient (covered by lane 7). Fractions containing the peak of MobMN199 were pooled, dialysed against buffer A and concentrated. M indicates the molecular weight standards (in kDa). ( c ) Purified MobMN199 was injected onto a gel filtration column, and its elution profile was recorded; the inset shows the SDS–PAGE gel with the purified protein and the molecular size markers. ( d ) Relaxation assays with wild-type MobM and the MobMN199 protein. Supercoiled pMV158 DNA samples (300 ng; 8 nM) were incubated with and without (−) full-length MobM (left) or with the short MobMN199 fragment (right) in the presence of 15 mM MnCl 2 at 30°C, 20 min. Protein concentrations used were 120, 240 and 480 nM. Generation of relaxed forms (FII) from supercoiled DNA forms (FI) was analysed by electrophoresis on 1% agarose gels without prior staining with EtBr, conditions in which forms FI′ are not resolved. The amounts of relaxed DNA forms generated by treatment of MobM and by MobMN199 were calculated by subtracting the amount of already nicked molecules (faint FII band in the untreated samples generated by mechanical shearing) from the FII-forms generated by protein treatment. The values of the protein-relaxed molecules were 25, 42 and 62%, and 28, 40 and 63% for samples treated with MobM and MobMN199, respectively. The weak band above relaxed forms FII has been observed before ( 20 ) and might correspond to relaxed DNA dimers.

Techniques Used: Purification, Electrophoresis, Molecular Weight, Injection, Filtration, SDS Page, Incubation, Staining, Generated

4) Product Images from "The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban"

Article Title: The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban

Journal: EMBO Reports

doi: 10.15252/embr.201846449

Analysis of  SPPL2c −/−  testis No change in SPP, SPPL2a and SPPL2b expression in testis of SPPL2c‐deficient mice. Total RNA was isolated from testis of wild‐type and  SPPL2c −/−  mice ( n  = 3). In the resulting cDNA,  SPP ,  SPPL2a  and  SPPL2b  transcript abundance was quantified by qRT–PCR and normalised to that of  Tuba1 a. Bars depict mean values normalised to those of wild‐type samples ± SD. Western Blot analysis of SPP protein in total lysates from wild‐type and  SPPL2c −/−  testis. To facilitate identification of murine endogenous SPP, HEK293 cells transiently expressing murine SPP carrying a C‐terminal Myc epitope or just transfected with empty vector (−) were analysed. Due to the strong expression in these cells, only 1/8 of the protein amount was loaded from these samples as compared to the testis lysates. In addition to the SPP monomer (open arrowhead), we predominantly detected dimeric SPP (closed arrowhead), which exhibits a high stability under these experimental conditions. Actin was detected as control for protein loading. β‐galactosidase reporter expression in  SPPL2c −/−  testis. Total lysates from wild‐type and  SPPL2c −/−  testis were analysed by Western blotting for  E. coli  β‐galactosidase, SPPL2c and Actin. Gating strategy for sorting of individual germ cell populations based on their DNA content as determined by Hoechst 33342 staining. Cells were first roughly gated based on their forward (FSC) and sideward scatter (SSC) prior to exclusion of PI‐positive dead cells. Finally, 1C (haploid cells including spermatids), 2C (spermatogonia, secondary spermatocytes, Sertoli cells, other somatic cells) and 4C cells (primary spermatocytes, G2 spermatogonia) were gated based on their individual Hoechst staining as depicted. Immunohistochemical visualisation of SPP in paraffin sections from Bouin‐fixed wild‐type and  SPPL2c −/−  testis. Prior to immunostaining, sections were subjected to epitope retrieval in citrate buffer. A polyclonal SPP antiserum generated against an internal epitope of murine SPP was used as primary antibody. As a control for antibody specificity, stainings with normal rabbit polyclonal IgG were performed in parallel. Scale bars, 100 μm. The reduction of SPPL2c‐deficient spermatids is not caused by apoptosis. TUNEL staining was performed on paraffin sections from wild‐type and  SPPL2c −/−  testis ( n  = 3 per genotype), as shown here from a representative example. Labelled apoptotic cells were observed among the spermatogonia (arrow) and primary spermatocytes (arrowhead), however at a similar frequency in both genotypes. No relevant labelling was seen in spermatids neither in wild‐type nor in  SPPL2c −/−  sections. Scale bars, 100 μm. Source data are available online for this figure.
Figure Legend Snippet: Analysis of SPPL2c −/− testis No change in SPP, SPPL2a and SPPL2b expression in testis of SPPL2c‐deficient mice. Total RNA was isolated from testis of wild‐type and SPPL2c −/− mice ( n  = 3). In the resulting cDNA, SPP , SPPL2a and SPPL2b transcript abundance was quantified by qRT–PCR and normalised to that of Tuba1 a. Bars depict mean values normalised to those of wild‐type samples ± SD. Western Blot analysis of SPP protein in total lysates from wild‐type and SPPL2c −/− testis. To facilitate identification of murine endogenous SPP, HEK293 cells transiently expressing murine SPP carrying a C‐terminal Myc epitope or just transfected with empty vector (−) were analysed. Due to the strong expression in these cells, only 1/8 of the protein amount was loaded from these samples as compared to the testis lysates. In addition to the SPP monomer (open arrowhead), we predominantly detected dimeric SPP (closed arrowhead), which exhibits a high stability under these experimental conditions. Actin was detected as control for protein loading. β‐galactosidase reporter expression in SPPL2c −/− testis. Total lysates from wild‐type and SPPL2c −/− testis were analysed by Western blotting for E. coli β‐galactosidase, SPPL2c and Actin. Gating strategy for sorting of individual germ cell populations based on their DNA content as determined by Hoechst 33342 staining. Cells were first roughly gated based on their forward (FSC) and sideward scatter (SSC) prior to exclusion of PI‐positive dead cells. Finally, 1C (haploid cells including spermatids), 2C (spermatogonia, secondary spermatocytes, Sertoli cells, other somatic cells) and 4C cells (primary spermatocytes, G2 spermatogonia) were gated based on their individual Hoechst staining as depicted. Immunohistochemical visualisation of SPP in paraffin sections from Bouin‐fixed wild‐type and SPPL2c −/− testis. Prior to immunostaining, sections were subjected to epitope retrieval in citrate buffer. A polyclonal SPP antiserum generated against an internal epitope of murine SPP was used as primary antibody. As a control for antibody specificity, stainings with normal rabbit polyclonal IgG were performed in parallel. Scale bars, 100 μm. The reduction of SPPL2c‐deficient spermatids is not caused by apoptosis. TUNEL staining was performed on paraffin sections from wild‐type and SPPL2c −/− testis ( n  = 3 per genotype), as shown here from a representative example. Labelled apoptotic cells were observed among the spermatogonia (arrow) and primary spermatocytes (arrowhead), however at a similar frequency in both genotypes. No relevant labelling was seen in spermatids neither in wild‐type nor in SPPL2c −/− sections. Scale bars, 100 μm. Source data are available online for this figure.

Techniques Used: Expressing, Mouse Assay, Isolation, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Staining, Immunohistochemistry, Immunostaining, Generated, TUNEL Assay

5) Product Images from "Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation"

Article Title: Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation

Journal: Genes & Development

doi: 10.1101/gad.1093903

Mcl-1 and Bcl-x L are necessary and sufficient for cytosolic inhibitory activity. ( A ) Mcl-1 and Bcl-x L correlate with inhibitory activity after ammonium sulfate fractionation. Ammonium sulfate was added to 1 mL of S100 (5 mg/mL) up to 30% (lanes 3,4 ) or 50% (lanes 5,6 ). Following incubation at 4°C for 1 h, the supernatant (Sup) and pellet (Pel) were separated by centrifugation (20,000 g ). The pellet was resuspended in 1 mL of Buffer A. Both the supernatants and pellets were dialyzed in Buffer A overnight. All fractions were assayed for inhibitory activity (as described in Materials and Methods) and evaluated for Mcl-1 and Bcl-x L . ( B ) HeLa S100 (36 mg) was first precipitated with 30% ammonium sulfate. The resulting pellet (7.5 mg) was resuspended and dialyzed in Buffer A and then loaded onto a 1-mL Hi-trap Q Sepharose column (Amersham) equilibrated in Buffer A. The protein was eluted with a gradient from 0 to 750 mM NaCl (in Buffer A) over 14 mL. Inhibitory activity was assayed for buffer alone (lane 1 ), input (lane 2 ), Q flow through (lane 3 ), and fractions eluting from Q sepharose (lanes 4 - 13 ). The amount of Mcl-1 and Bcl-x L in each sample and the mitochondria (lane 1 ) was measured by Western blot. ( C ) HeLa S100 was immunodepleted as described in Materials and Methods. Inhibitory activity was assayed in buffer alone (lane 1 ), S100 mock-immunodepleted (lane 2 ), depleted of Mcl-1 (lane 3 ), Bcl-x L (lane 4 ), or both (lane 5 ). The amount of Mcl-1 and Bcl-x L was determined by Western blot for each S100 sample. ( D ) Recombinant Mcl-1 (rMcl-1) and Bcl-x L (rBcl-x L ) were prepared as described in Materials and Methods. S100, rMcl-1, and rBcl-x L were analyzed for inhibitory activity. The levels of Mcl-1 and Bcl-x L in recombinant fractions were compared with those in S100. Mitochondria solubilized in Buffer A with 1% NP-40 were analyzed for Mcl-1 and Bcl-x L to compare the levels of these proteins in mitochondria to those in the fractions.
Figure Legend Snippet: Mcl-1 and Bcl-x L are necessary and sufficient for cytosolic inhibitory activity. ( A ) Mcl-1 and Bcl-x L correlate with inhibitory activity after ammonium sulfate fractionation. Ammonium sulfate was added to 1 mL of S100 (5 mg/mL) up to 30% (lanes 3,4 ) or 50% (lanes 5,6 ). Following incubation at 4°C for 1 h, the supernatant (Sup) and pellet (Pel) were separated by centrifugation (20,000 g ). The pellet was resuspended in 1 mL of Buffer A. Both the supernatants and pellets were dialyzed in Buffer A overnight. All fractions were assayed for inhibitory activity (as described in Materials and Methods) and evaluated for Mcl-1 and Bcl-x L . ( B ) HeLa S100 (36 mg) was first precipitated with 30% ammonium sulfate. The resulting pellet (7.5 mg) was resuspended and dialyzed in Buffer A and then loaded onto a 1-mL Hi-trap Q Sepharose column (Amersham) equilibrated in Buffer A. The protein was eluted with a gradient from 0 to 750 mM NaCl (in Buffer A) over 14 mL. Inhibitory activity was assayed for buffer alone (lane 1 ), input (lane 2 ), Q flow through (lane 3 ), and fractions eluting from Q sepharose (lanes 4 - 13 ). The amount of Mcl-1 and Bcl-x L in each sample and the mitochondria (lane 1 ) was measured by Western blot. ( C ) HeLa S100 was immunodepleted as described in Materials and Methods. Inhibitory activity was assayed in buffer alone (lane 1 ), S100 mock-immunodepleted (lane 2 ), depleted of Mcl-1 (lane 3 ), Bcl-x L (lane 4 ), or both (lane 5 ). The amount of Mcl-1 and Bcl-x L was determined by Western blot for each S100 sample. ( D ) Recombinant Mcl-1 (rMcl-1) and Bcl-x L (rBcl-x L ) were prepared as described in Materials and Methods. S100, rMcl-1, and rBcl-x L were analyzed for inhibitory activity. The levels of Mcl-1 and Bcl-x L in recombinant fractions were compared with those in S100. Mitochondria solubilized in Buffer A with 1% NP-40 were analyzed for Mcl-1 and Bcl-x L to compare the levels of these proteins in mitochondria to those in the fractions.

Techniques Used: Activity Assay, Fractionation, Incubation, Centrifugation, Flow Cytometry, Western Blot, Recombinant

6) Product Images from "A novel Apaf-1-independent putative caspase-2 activation complex"

Article Title: A novel Apaf-1-independent putative caspase-2 activation complex

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200209004

Caspase-2 is recruited to a large protein complex. HeLa cell lysates were subjected to size exclusion chromatography (A and B). Aliquots from the fractions were analyzed by immunoblotting using the monoclonal antibody 11B4 that recognizes the p19 subunit and full-length caspase-2. Cells were lysed in buffer A and incubated at 4 or 37°C for 60 min before loading onto the column. In B, lysates were incubated at 4 or 37°C for 2 h with 2 μg cytochrome c and 2 mM dATP. The elution positions of the markers on the Superdex 200 column are indicated. The positions of the SDS-PAGE prestained M r standards are indicated on the right of the blots. The smaller caspase-2 immunoreactive band is prodomain+p19 processing intermediate that is often detected in cell lysates, probably due to some processing of procaspase-2 during cell harvesting and lysis.
Figure Legend Snippet: Caspase-2 is recruited to a large protein complex. HeLa cell lysates were subjected to size exclusion chromatography (A and B). Aliquots from the fractions were analyzed by immunoblotting using the monoclonal antibody 11B4 that recognizes the p19 subunit and full-length caspase-2. Cells were lysed in buffer A and incubated at 4 or 37°C for 60 min before loading onto the column. In B, lysates were incubated at 4 or 37°C for 2 h with 2 μg cytochrome c and 2 mM dATP. The elution positions of the markers on the Superdex 200 column are indicated. The positions of the SDS-PAGE prestained M r standards are indicated on the right of the blots. The smaller caspase-2 immunoreactive band is prodomain+p19 processing intermediate that is often detected in cell lysates, probably due to some processing of procaspase-2 during cell harvesting and lysis.

Techniques Used: Size-exclusion Chromatography, Incubation, SDS Page, Cell Harvesting, Lysis

7) Product Images from "Exportin-5 mediates nuclear export of SRP RNA in vertebrates"

Article Title: Exportin-5 mediates nuclear export of SRP RNA in vertebrates

Journal: Genes to Cells

doi: 10.1111/gtc.12218

Exportin-5 interacts with SRP RNA. (A) Biotinylated human SRP RNA was incubated with HeLa cell nuclear extracts (HNE) with or without RanQ69LGTP in buffer A for 30 min at 30°C, and then biotinylated human SRP RNA was pulled down by streptavidin beads. The precipitated proteins were analyzed by Western blotting. (B) The same as A except that the nonbiotinylated RNA competitors were used. (C) The pull-down assay using biotinylated human SRP RNA, the recombinant Exportin-5 and RanQ69LGTP was similarly carried out as A. The precipitated Exportin-5 was analyzed by Western blotting using anti-His tag antibody.
Figure Legend Snippet: Exportin-5 interacts with SRP RNA. (A) Biotinylated human SRP RNA was incubated with HeLa cell nuclear extracts (HNE) with or without RanQ69LGTP in buffer A for 30 min at 30°C, and then biotinylated human SRP RNA was pulled down by streptavidin beads. The precipitated proteins were analyzed by Western blotting. (B) The same as A except that the nonbiotinylated RNA competitors were used. (C) The pull-down assay using biotinylated human SRP RNA, the recombinant Exportin-5 and RanQ69LGTP was similarly carried out as A. The precipitated Exportin-5 was analyzed by Western blotting using anti-His tag antibody.

Techniques Used: Incubation, Western Blot, Pull Down Assay, Recombinant

8) Product Images from "Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases"

Article Title: Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200412071

Cdk5 phosphorylates htt in vitro and in vivo. (A) GST and GST-tagged htt1-588 (GST-htt588) (wild-type) were purified from E. coli . Both proteins were phosphorylated by 0.1 μg of p35–cdk5 complexes. Top panel shows phosphorylated GST (lane 1) and GST-htt588 (lane 2). Bottom panel shows purified GST (lane 1) and GST-htt588 (lane 2). (B) p35–cdk5 was cotransfected to COS-7 cells. We immunoprecipitated p35–cdk5 with anti-cdk5. Httwt588 or httmu588 were pulled down with anti-Flag from distinct COS-7 cells transfected with these constructs. The figure shows in vitro kinase assays (top) and anti-Flag blot (bottom) from p35–cdk5 incubated with httwt588 and γ-[ 32 P]ATP (lane 1) and p35–cdk5 incubated with httmu588 and γ-[ 32 P]ATP (lane 2). The mixtures were resolved with 10% SDS-PAGE, and then transferred to PVDF membrane and subjected to autoradiography (top). The PVDF membrane was blotted with anti-Flag (bottom). (C) PC-12 cells were starved for 24 h, and then induced to differentiate with 100 ng/ml NGF for 48 h. Cells were treated with 20 μM of the cdk5 inhibitor roscovitine (Rosco) or DMSO (control) when cells were induced to differentiate with NGF. After 48 h of treatment, PC-12 cells were lysed in buffer A containing phosphatase inhibitors. Htt was immunoprecipitated with anti-htt antibody, 2166, and then detected with the antiphosphoserine antibody 16B4 (top) and anti-htt (middle). p35–cdk5 complex was pulled down from the differentiated PC-12 cells and an in vitro kinase assay was performed in the presence of DMSO (lane 1) or roscovitine (lane 2) using histone H1 as a substrate (bottom).
Figure Legend Snippet: Cdk5 phosphorylates htt in vitro and in vivo. (A) GST and GST-tagged htt1-588 (GST-htt588) (wild-type) were purified from E. coli . Both proteins were phosphorylated by 0.1 μg of p35–cdk5 complexes. Top panel shows phosphorylated GST (lane 1) and GST-htt588 (lane 2). Bottom panel shows purified GST (lane 1) and GST-htt588 (lane 2). (B) p35–cdk5 was cotransfected to COS-7 cells. We immunoprecipitated p35–cdk5 with anti-cdk5. Httwt588 or httmu588 were pulled down with anti-Flag from distinct COS-7 cells transfected with these constructs. The figure shows in vitro kinase assays (top) and anti-Flag blot (bottom) from p35–cdk5 incubated with httwt588 and γ-[ 32 P]ATP (lane 1) and p35–cdk5 incubated with httmu588 and γ-[ 32 P]ATP (lane 2). The mixtures were resolved with 10% SDS-PAGE, and then transferred to PVDF membrane and subjected to autoradiography (top). The PVDF membrane was blotted with anti-Flag (bottom). (C) PC-12 cells were starved for 24 h, and then induced to differentiate with 100 ng/ml NGF for 48 h. Cells were treated with 20 μM of the cdk5 inhibitor roscovitine (Rosco) or DMSO (control) when cells were induced to differentiate with NGF. After 48 h of treatment, PC-12 cells were lysed in buffer A containing phosphatase inhibitors. Htt was immunoprecipitated with anti-htt antibody, 2166, and then detected with the antiphosphoserine antibody 16B4 (top) and anti-htt (middle). p35–cdk5 complex was pulled down from the differentiated PC-12 cells and an in vitro kinase assay was performed in the presence of DMSO (lane 1) or roscovitine (lane 2) using histone H1 as a substrate (bottom).

Techniques Used: In Vitro, In Vivo, Purification, Immunoprecipitation, Transfection, Construct, Incubation, SDS Page, Autoradiography, Kinase Assay

9) Product Images from "Palytoxin and an Ostreopsis Toxin Extract Increase the Levels of mRNAs Encoding Inflammation-Related Proteins in Human Macrophages via p38 MAPK and NF-?B"

Article Title: Palytoxin and an Ostreopsis Toxin Extract Increase the Levels of mRNAs Encoding Inflammation-Related Proteins in Human Macrophages via p38 MAPK and NF-?B

Journal: PLoS ONE

doi: 10.1371/journal.pone.0038139

IκBα and p65 protein levels in cells treated with the vehicle, the OSTRTX extract and PLTX. ( A ) IκBα protein levels were determined in whole cell lysates obtained from macrophages incubated 4 h with the vehicle (lane 1), the OSTRTX extract (lanes 2–3) or PLTX (lane 4). Protein extracts (15 µg) were resolved by SDS-PAGE on 8% gel and then submitted to Western immunoblotting. Blots were probed with an anti-IκBα (upper panel) and an anti-p65 (RelA) antibody to check for protein loading (lower panel). ( B ) intracellular content and subcellular distribution of p65 (RelA) in PLTX- versus vehicle-treated cells was assessed by immunoblotting analysis of whole (15 µg, lanes 1–2), cytosolic (15 µg, lanes 3–4) and nuclear (10 µg, lane 5–6) extracts. Whole cell extracts were obtained by lysing cells in SDS buffer. In parallel, cells were sub-fractionated by extraction in Buffer A (BUFF A, cytosolic proteins) followed by Buffer B (nuclear proteins, BUFF B), the residual material, containing insoluble proteins, including those associated with cytoskeletal structures, was solubilized in SDS-PAGE sample buffer and run in parallel (lanes 7–8). ( C ) Approaches to inhibit p65 in vitro degradation during extraction in native conditions. Cell pellets, containing an equivalent number of macrophages, were lysed in SDS buffer (SDS BUFF, lane 1), Buffer A (BUFF A, lane 2) or Buffer A supplemented with palytoxin (BUFF A+PLTX, lane 3). An equal volume of SDS sample buffer was added to all tubes and comparable volumes of the resulting protein extracts were resolved by electrophoresis and immunoblotted with an anti p65 antibody.( D ) cell pellets as in C were lysed in SDS buffer (lane 1), Buffer A (lane 2) and Buffer A further supplemented with lysososmal protease inhibitors (BUFF A PLUS, lane 3) and submitted to SDS-PAGE and immunoblotting with an anti p65 antibody.
Figure Legend Snippet: IκBα and p65 protein levels in cells treated with the vehicle, the OSTRTX extract and PLTX. ( A ) IκBα protein levels were determined in whole cell lysates obtained from macrophages incubated 4 h with the vehicle (lane 1), the OSTRTX extract (lanes 2–3) or PLTX (lane 4). Protein extracts (15 µg) were resolved by SDS-PAGE on 8% gel and then submitted to Western immunoblotting. Blots were probed with an anti-IκBα (upper panel) and an anti-p65 (RelA) antibody to check for protein loading (lower panel). ( B ) intracellular content and subcellular distribution of p65 (RelA) in PLTX- versus vehicle-treated cells was assessed by immunoblotting analysis of whole (15 µg, lanes 1–2), cytosolic (15 µg, lanes 3–4) and nuclear (10 µg, lane 5–6) extracts. Whole cell extracts were obtained by lysing cells in SDS buffer. In parallel, cells were sub-fractionated by extraction in Buffer A (BUFF A, cytosolic proteins) followed by Buffer B (nuclear proteins, BUFF B), the residual material, containing insoluble proteins, including those associated with cytoskeletal structures, was solubilized in SDS-PAGE sample buffer and run in parallel (lanes 7–8). ( C ) Approaches to inhibit p65 in vitro degradation during extraction in native conditions. Cell pellets, containing an equivalent number of macrophages, were lysed in SDS buffer (SDS BUFF, lane 1), Buffer A (BUFF A, lane 2) or Buffer A supplemented with palytoxin (BUFF A+PLTX, lane 3). An equal volume of SDS sample buffer was added to all tubes and comparable volumes of the resulting protein extracts were resolved by electrophoresis and immunoblotted with an anti p65 antibody.( D ) cell pellets as in C were lysed in SDS buffer (lane 1), Buffer A (lane 2) and Buffer A further supplemented with lysososmal protease inhibitors (BUFF A PLUS, lane 3) and submitted to SDS-PAGE and immunoblotting with an anti p65 antibody.

Techniques Used: Incubation, SDS Page, Western Blot, In Vitro, Electrophoresis

10) Product Images from "Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis ▿"

Article Title: Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.00443-10

Recombinant N-His 6 -GpsA protein is an active G3P dehydrogenase  in vitro. R. prowazekii gpsA  was cloned into pET15b, heterologously expressed in  E. coli  BL21(DE3), and purified with N-His 6 -GpsA. (A) Purified protein was resolved on a 4 to 15% Tris-HCl SDS-polyacrylamide gel and visualized by Imperial protein staining and Western blot analysis using anti-His 6  monoclonal antibody (sizes [in kDa] are indicated on the left). The mass spectrometry analysis coverage map shows matched peptides in bold font (30% coverage). (B) N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P was verified by paper chromatography. [ 32 P]ATP, [ 32 P]DHAP, [ 32 P]G3P, and [ 32 P]orthophosphate were used as standards. The reaction mixtures were incubated for 60 min at room temperature prior to chromatographic analysis. Lane A, a negative-control reaction mixture containing 6 μM [ 32 P]DHAP and 100 μM NADPH only; lane B, a negative-control reaction mixture containing [ 32 P]DHAP and 2.5 μg ml −1  N-His 6 -GpsA protein only; lane C, the N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P absolutely required the presence of NADPH. The density of each compound on the chromatograph is expressed as a percentage of the total lane density (summarized in tabular form to the right of each spot on the chromatograph). (C) N-His 6 -GpsA-catalyzed conversion of DHAP to G3P was measured spectrophotometrically at OD 340  by following the oxidation of NADPH or NADH over time. Error bars represent standard deviations. The reaction mixtures contained 1.0 μg ml −1  N-His 6 -GpsA, 500 μM DHAP, and 100 μM NAD(P)H in modified buffer A; and the reactions were carried out at room temperature.
Figure Legend Snippet: Recombinant N-His 6 -GpsA protein is an active G3P dehydrogenase in vitro. R. prowazekii gpsA was cloned into pET15b, heterologously expressed in E. coli BL21(DE3), and purified with N-His 6 -GpsA. (A) Purified protein was resolved on a 4 to 15% Tris-HCl SDS-polyacrylamide gel and visualized by Imperial protein staining and Western blot analysis using anti-His 6 monoclonal antibody (sizes [in kDa] are indicated on the left). The mass spectrometry analysis coverage map shows matched peptides in bold font (30% coverage). (B) N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P was verified by paper chromatography. [ 32 P]ATP, [ 32 P]DHAP, [ 32 P]G3P, and [ 32 P]orthophosphate were used as standards. The reaction mixtures were incubated for 60 min at room temperature prior to chromatographic analysis. Lane A, a negative-control reaction mixture containing 6 μM [ 32 P]DHAP and 100 μM NADPH only; lane B, a negative-control reaction mixture containing [ 32 P]DHAP and 2.5 μg ml −1 N-His 6 -GpsA protein only; lane C, the N-His 6 -GpsA-catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P absolutely required the presence of NADPH. The density of each compound on the chromatograph is expressed as a percentage of the total lane density (summarized in tabular form to the right of each spot on the chromatograph). (C) N-His 6 -GpsA-catalyzed conversion of DHAP to G3P was measured spectrophotometrically at OD 340 by following the oxidation of NADPH or NADH over time. Error bars represent standard deviations. The reaction mixtures contained 1.0 μg ml −1 N-His 6 -GpsA, 500 μM DHAP, and 100 μM NAD(P)H in modified buffer A; and the reactions were carried out at room temperature.

Techniques Used: Recombinant, In Vitro, Clone Assay, Purification, Staining, Western Blot, Mass Spectrometry, Paper Chromatography, Incubation, Negative Control, Modification

G3PDH activity is detectable in  R. prowazekii  lysed-cell extracts. Hen egg yolk sac-purified  R. prowazekii  cells were lysed by ballistic shearing, and the lysed-cell extracts were assayed for G3PDH activity. (A) Rickettsial lysed-cell extracts catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P, as determined by paper chromatography. [ 32 P]ATP, [ 32 P]DHAP, [ 32 P]G3P, and [ 32 P]orthophosphate were used as standards. Lane A, a negative-control (-ve Ctrl) reaction mixture containing 75 μM [ 32 P]DHAP and 100 μM NADPH only; lane B, a reaction mixture containing [ 32 P]DHAP, NADPH, and 0.5 mg ml −1  of rickettsial lysed-cell extract; lane C, a reaction mixture containing [ 32 P]DHAP, NADPH, and 1 mg ml −1  of rickettsial lysed-cell extract; lane D, a reaction mixture containing [ 32 P]DHAP, NADPH, and 2 mg ml −1  of rickettsial lysed-cell extract. The density of each compound on the chromatograph is expressed as a percentage of the total lane density (summarized in tabular form to the right of the chromatograph). (B) Rickettsial lysed-cell extract-catalyzed conversion of DHAP to G3P was measured spectrophotometrically at an OD 340  by following the oxidation of NADPH or NADH. Error bars represent standard deviations. The reaction mixtures contained 1.4 mg ml −1  of rickettsial lysed-cell extract, 500 μM DHAP, and 100 μM NAD(P)H in modified buffer A; and the reactions were carried out at room temperature.
Figure Legend Snippet: G3PDH activity is detectable in R. prowazekii lysed-cell extracts. Hen egg yolk sac-purified R. prowazekii cells were lysed by ballistic shearing, and the lysed-cell extracts were assayed for G3PDH activity. (A) Rickettsial lysed-cell extracts catalyzed conversion of [ 32 P]DHAP to [ 32 P]G3P, as determined by paper chromatography. [ 32 P]ATP, [ 32 P]DHAP, [ 32 P]G3P, and [ 32 P]orthophosphate were used as standards. Lane A, a negative-control (-ve Ctrl) reaction mixture containing 75 μM [ 32 P]DHAP and 100 μM NADPH only; lane B, a reaction mixture containing [ 32 P]DHAP, NADPH, and 0.5 mg ml −1 of rickettsial lysed-cell extract; lane C, a reaction mixture containing [ 32 P]DHAP, NADPH, and 1 mg ml −1 of rickettsial lysed-cell extract; lane D, a reaction mixture containing [ 32 P]DHAP, NADPH, and 2 mg ml −1 of rickettsial lysed-cell extract. The density of each compound on the chromatograph is expressed as a percentage of the total lane density (summarized in tabular form to the right of the chromatograph). (B) Rickettsial lysed-cell extract-catalyzed conversion of DHAP to G3P was measured spectrophotometrically at an OD 340 by following the oxidation of NADPH or NADH. Error bars represent standard deviations. The reaction mixtures contained 1.4 mg ml −1 of rickettsial lysed-cell extract, 500 μM DHAP, and 100 μM NAD(P)H in modified buffer A; and the reactions were carried out at room temperature.

Techniques Used: Activity Assay, Purification, Paper Chromatography, Negative Control, Modification

11) Product Images from "Complicated behavior of G-quadruplexes and evaluating G-quadruplexes' ligands in various systems mimicking cellular circumstance"

Article Title: Complicated behavior of G-quadruplexes and evaluating G-quadruplexes' ligands in various systems mimicking cellular circumstance

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2015.09.022

Effects of different systems on the polymerase stop assay with HTG21 and its corresponding mutant sequence HTG21-mu in a normal PCR reaction buffer, the cell free system,  Xenopus laevis  egg extract, dialysis buffer, dialysis buffer A, and control system mentioned before. Data represented means of six independent experiments with standard error.
Figure Legend Snippet: Effects of different systems on the polymerase stop assay with HTG21 and its corresponding mutant sequence HTG21-mu in a normal PCR reaction buffer, the cell free system, Xenopus laevis egg extract, dialysis buffer, dialysis buffer A, and control system mentioned before. Data represented means of six independent experiments with standard error.

Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction

12) Product Images from "Characterization of the L-Lactate Dehydrogenase from Aggregatibacter actinomycetemcomitans"

Article Title: Characterization of the L-Lactate Dehydrogenase from Aggregatibacter actinomycetemcomitans

Journal: PLoS ONE

doi: 10.1371/journal.pone.0007864

Purification of LctD-his 6 . (A) SDS-PAGE analysis of LctD-his 6 . LctD-his 6 was purified using a HisTrap nickel column and examined by SDS-PAGE and Coomassie staining. Lane designations above the gel are: (La), molecular weight ladder; (Ly), cell lysate; (W1), buffer A flow-through; (W2), buffer B with 0.15 M imidazole flow-through; (W3), buffer B with 0.5 M imidazole flow-through; (LctD), phosphate buffer-exchanged LctD-his 6 . Numbers to the left of the SDS-PAGE gel represent size standards in kilodaltons. Phosphate buffer-exchanged LctD-his 6 was used for enzymatic activity studies. (B) Western blot analysis of purified LctD-his 6 . Purified LctD-his 6 was separated on a 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and detected using an anti-his 6 antibody and chemiluminescence. Image represents an overlay of a white light image and a chemiluminescent image.
Figure Legend Snippet: Purification of LctD-his 6 . (A) SDS-PAGE analysis of LctD-his 6 . LctD-his 6 was purified using a HisTrap nickel column and examined by SDS-PAGE and Coomassie staining. Lane designations above the gel are: (La), molecular weight ladder; (Ly), cell lysate; (W1), buffer A flow-through; (W2), buffer B with 0.15 M imidazole flow-through; (W3), buffer B with 0.5 M imidazole flow-through; (LctD), phosphate buffer-exchanged LctD-his 6 . Numbers to the left of the SDS-PAGE gel represent size standards in kilodaltons. Phosphate buffer-exchanged LctD-his 6 was used for enzymatic activity studies. (B) Western blot analysis of purified LctD-his 6 . Purified LctD-his 6 was separated on a 10% SDS-PAGE gel, transferred to nitrocellulose membrane, and detected using an anti-his 6 antibody and chemiluminescence. Image represents an overlay of a white light image and a chemiluminescent image.

Techniques Used: Purification, SDS Page, Nickel Column, Staining, Molecular Weight, Flow Cytometry, Activity Assay, Western Blot

13) Product Images from "Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles"

Article Title: Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles

Journal: Biochemical Journal

doi: 10.1042/BJ20131540

Characterization of purified Erv1 mutant proteins ( a ) Gel-filtration profiles of Erv1SXXC (black curve) and Erv1 CXXS (grey curve) compared with that of WT (dotted curve) the corresponding double cysteine mutant Erv1 SXXS (dashed curve) on Superdex 75 column equilibrated with buffer A [50 mM Tris/HCl buffer (pH 7.4) and 150 mM NaCl]. The molecular mass and the corresponding peak fractions are indicated. ( b ) UV–visible absorption spectra of the Erv1 SXXC and Erv1 CXXS mutant proteins. Peak fractions separated by size-exclusion chromatography in ( a ) were analysed: Erv1 SXXC peak 2 (black curve), Erv1 SXXC peak 1 (dashed curve), Erv1 CXXS peak 1 (grey curve) and Erv1 SXXC peak 2 treated with 2 mM of immobilized TCEP (dotted curve). All measurements were done in buffer A at 25°C.
Figure Legend Snippet: Characterization of purified Erv1 mutant proteins ( a ) Gel-filtration profiles of Erv1SXXC (black curve) and Erv1 CXXS (grey curve) compared with that of WT (dotted curve) the corresponding double cysteine mutant Erv1 SXXS (dashed curve) on Superdex 75 column equilibrated with buffer A [50 mM Tris/HCl buffer (pH 7.4) and 150 mM NaCl]. The molecular mass and the corresponding peak fractions are indicated. ( b ) UV–visible absorption spectra of the Erv1 SXXC and Erv1 CXXS mutant proteins. Peak fractions separated by size-exclusion chromatography in ( a ) were analysed: Erv1 SXXC peak 2 (black curve), Erv1 SXXC peak 1 (dashed curve), Erv1 CXXS peak 1 (grey curve) and Erv1 SXXC peak 2 treated with 2 mM of immobilized TCEP (dotted curve). All measurements were done in buffer A at 25°C.

Techniques Used: Purification, Mutagenesis, Filtration, Size-exclusion Chromatography

Cys 30 is more reactive than Cys 33 for intermolecular disulfide bond formation ( a ) Tris/Tricine SDS/PAGE (16% gel) of Erv1 SXXC and Erv1 CXXS mutant protein peak fractions in Figure 5 (a) under reducing and non-reducing conditions. ( b ) Mixed disulfide bond formation between single cysteine mutants of Mia40 CPC (Mia40 SPC and Mia40 CPS ) and Erv1 shuttle cysteine residues (Erv1 SXXC and Erv1 CXXS ). The mutant proteins were pre-treated briefly with 2 mM TCEP and buffer-exchanged to buffer A before incubation at equimolar concentrations of 5 μM for 20 min at room temperature. The reactions were stopped by the addition of sample buffer with 1 mM DTT or 2.5 mM AMS. The proteins were detected by Western blotting with an antibody against Erv1. ( c ) Time courses of mixed disulfide bond formation between Mia40 and the Erv1 mutants. The proteins were treated as in ( b ) over a 10-min incubation. The molar ratio concentration of Mia40 to Erv1 used was 10:1 (50 μM Mia40 and 5 μM Erv1). The reactions were stopped at each time point by addition of sample buffer containing 20 mM IAM. As a reducing control, reactions at the end of each time course were analysed under reducing conditions by resuspending the reaction mixtures in sample buffer containing DTT.
Figure Legend Snippet: Cys 30 is more reactive than Cys 33 for intermolecular disulfide bond formation ( a ) Tris/Tricine SDS/PAGE (16% gel) of Erv1 SXXC and Erv1 CXXS mutant protein peak fractions in Figure 5 (a) under reducing and non-reducing conditions. ( b ) Mixed disulfide bond formation between single cysteine mutants of Mia40 CPC (Mia40 SPC and Mia40 CPS ) and Erv1 shuttle cysteine residues (Erv1 SXXC and Erv1 CXXS ). The mutant proteins were pre-treated briefly with 2 mM TCEP and buffer-exchanged to buffer A before incubation at equimolar concentrations of 5 μM for 20 min at room temperature. The reactions were stopped by the addition of sample buffer with 1 mM DTT or 2.5 mM AMS. The proteins were detected by Western blotting with an antibody against Erv1. ( c ) Time courses of mixed disulfide bond formation between Mia40 and the Erv1 mutants. The proteins were treated as in ( b ) over a 10-min incubation. The molar ratio concentration of Mia40 to Erv1 used was 10:1 (50 μM Mia40 and 5 μM Erv1). The reactions were stopped at each time point by addition of sample buffer containing 20 mM IAM. As a reducing control, reactions at the end of each time course were analysed under reducing conditions by resuspending the reaction mixtures in sample buffer containing DTT.

Techniques Used: SDS Page, Mutagenesis, Incubation, Affinity Magnetic Separation, Western Blot, Concentration Assay

14) Product Images from "Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis"

Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200302084

ER stress induces Bax and Bak conformational changes and oligomerization at the ER. (A) ER stresses induce the conformational changes of Bax and Bak. HeLa, MCF7, and 293T cells were treated with thapsigargin (Thap; 2 μM) or tunicamycin (Tuni; 5 μg/ml) for 36 h. Cells were fixed in 0.25% paraformaldehyde in PBS for 5 min. Cells were incubated with a control antibody (mouse IgG1) and conformation-sensitive antibodies against Bax or Bak, followed by incubation with FITC-conjugated secondary antibody. (B) ER stress induces Bax oligomerization at the ER. Wild-type MEFs were treated with brefeldin A (BFA; 10 μg/ml), Thap (2 μM), or Tuni (10 μg/ml) for 24 h. Cells were resuspended in hypotonic buffer A and disrupted. 5 mM BMH cross-linking reagent was added to cross-link the oligomerized proteins. Cells were subjected to subcellular fractionation to obtain the HM and LM fractions. 20 μg of total protein was separated on a 4–12% gradient NuPAGE gel. A polyclonal anti-Bax antibody was used to detect Bax. COX IV and calnexin are shown as indicators of the purity of the fractionation and as loading controls.
Figure Legend Snippet: ER stress induces Bax and Bak conformational changes and oligomerization at the ER. (A) ER stresses induce the conformational changes of Bax and Bak. HeLa, MCF7, and 293T cells were treated with thapsigargin (Thap; 2 μM) or tunicamycin (Tuni; 5 μg/ml) for 36 h. Cells were fixed in 0.25% paraformaldehyde in PBS for 5 min. Cells were incubated with a control antibody (mouse IgG1) and conformation-sensitive antibodies against Bax or Bak, followed by incubation with FITC-conjugated secondary antibody. (B) ER stress induces Bax oligomerization at the ER. Wild-type MEFs were treated with brefeldin A (BFA; 10 μg/ml), Thap (2 μM), or Tuni (10 μg/ml) for 24 h. Cells were resuspended in hypotonic buffer A and disrupted. 5 mM BMH cross-linking reagent was added to cross-link the oligomerized proteins. Cells were subjected to subcellular fractionation to obtain the HM and LM fractions. 20 μg of total protein was separated on a 4–12% gradient NuPAGE gel. A polyclonal anti-Bax antibody was used to detect Bax. COX IV and calnexin are shown as indicators of the purity of the fractionation and as loading controls.

Techniques Used: Incubation, Fractionation

Bax and Bak are localized to the ER in addition to their mitochondrial location. (A) Wild-type and bax − / − bak − / − MEFs as well as HeLa cells were resuspended in hypotonic buffer A and disrupted. Subcellular fractionation was performed to obtain the fractions for cytosol (S-100), HM (mitochondria enriched), and LM (the ER enriched). 20 μg of total protein from each fraction was separated on a 4–12% gradient NuPAGE gel. Antibodies against Bax, Bak, calnexin, and Cox IV were used for immunoblotting. (B) Mouse liver was homogenized in buffer A and fractionated in sucrose gradient as described in the Materials and methods. Fractions from crude mitochondria and the 1.5/1.8 M sucrose interface were probed with indicated antibodies. (C) The LM fraction was not contaminated by mitochondrial outer membrane. Fractions of wild-type MEFs were probed with an anti-Tom40 antibody. (D) Immunoelectron microscopy of Bax (left) and Bak (right) in wild-type MEFs using 10-nm gold particles. The bottom panels are the enlargements of the boxed areas of the top images. The arrowheads point to the ER. mt, mitochondria.
Figure Legend Snippet: Bax and Bak are localized to the ER in addition to their mitochondrial location. (A) Wild-type and bax − / − bak − / − MEFs as well as HeLa cells were resuspended in hypotonic buffer A and disrupted. Subcellular fractionation was performed to obtain the fractions for cytosol (S-100), HM (mitochondria enriched), and LM (the ER enriched). 20 μg of total protein from each fraction was separated on a 4–12% gradient NuPAGE gel. Antibodies against Bax, Bak, calnexin, and Cox IV were used for immunoblotting. (B) Mouse liver was homogenized in buffer A and fractionated in sucrose gradient as described in the Materials and methods. Fractions from crude mitochondria and the 1.5/1.8 M sucrose interface were probed with indicated antibodies. (C) The LM fraction was not contaminated by mitochondrial outer membrane. Fractions of wild-type MEFs were probed with an anti-Tom40 antibody. (D) Immunoelectron microscopy of Bax (left) and Bak (right) in wild-type MEFs using 10-nm gold particles. The bottom panels are the enlargements of the boxed areas of the top images. The arrowheads point to the ER. mt, mitochondria.

Techniques Used: Fractionation, Immuno-Electron Microscopy

15) Product Images from "Intramembrane Aromatic Interactions Influence the Lipid Sensitivities of Pentameric Ligand-gated Ion Channels *"

Article Title: Intramembrane Aromatic Interactions Influence the Lipid Sensitivities of Pentameric Ligand-gated Ion Channels *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.624395

Secondary structure and biophysical properties of ELIC exposed to low ionic strength buffer. A , infrared spectra showing aso-ELIC exchanged into 2 H 2 O Buffer A ( i ) or 2 H 2 O phosphate buffer (no added salt) ( ii ). The amide I bands both before ( gray ) and
Figure Legend Snippet: Secondary structure and biophysical properties of ELIC exposed to low ionic strength buffer. A , infrared spectra showing aso-ELIC exchanged into 2 H 2 O Buffer A ( i ) or 2 H 2 O phosphate buffer (no added salt) ( ii ). The amide I bands both before ( gray ) and

Techniques Used: Allele-specific Oligonucleotide

16) Product Images from "Complicated behavior of G-quadruplexes and evaluating G-quadruplexes' ligands in various systems mimicking cellular circumstance"

Article Title: Complicated behavior of G-quadruplexes and evaluating G-quadruplexes' ligands in various systems mimicking cellular circumstance

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2015.09.022

Effects of different systems on the polymerase stop assay with HTG21 and its corresponding mutant sequence HTG21-mu in a normal PCR reaction buffer, the cell free system,  Xenopus laevis  egg extract, dialysis buffer, dialysis buffer A, and control system mentioned before. Data represented means of six independent experiments with standard error.
Figure Legend Snippet: Effects of different systems on the polymerase stop assay with HTG21 and its corresponding mutant sequence HTG21-mu in a normal PCR reaction buffer, the cell free system, Xenopus laevis egg extract, dialysis buffer, dialysis buffer A, and control system mentioned before. Data represented means of six independent experiments with standard error.

Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction

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Article Snippet: Immunoprecipitation (IP) IP was performed using buffer A (20 mM Tris-HCl, pH 7.2, 2 mM MgCl2 , 150 mM NaCl, 5 mM NaF, 1 mM Na3 VO4 , 0.5% NP-40, and protease inhibitor cocktail [Roche]). .. Cells were lysed in buffer A for 20 min on ice, followed by centrifugation at 13,000 g for 15 min. Primary antibodies (or anti-Flag M2-agarose affinity gel and anti-cdk5 (J3)–coupling gel [Pierce Chemical Co.]) were added to a final concentration of 5 μg/ml and incubated for 2 h to overnight at 4°C.

Article Title: ABIN-2 Forms a Ternary Complex with TPL-2 and NF-?B1 p105 and Is Essential for TPL-2 Protein Stability †
Article Snippet: Cell pellets (20 ml) of C3.25 HA-p105(S927A) and EV HeLa S3 cells were prepared by centrifugation from large-scale suspension cell cultures (20 liters). .. Cells were lysed in 10 volumes of ice-cold buffer A (1% Nonidet P-40 [NP-40], 50 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 1 mM Na3 VO4 , 1 mM Na4 P2 O7 plus a mixture of protease inhibitors [Roche Molecular Biochemicals]).

Article Title: Palytoxin and an Ostreopsis Toxin Extract Increase the Levels of mRNAs Encoding Inflammation-Related Proteins in Human Macrophages via p38 MAPK and NF-?B
Article Snippet: After treatment, cells were extensively washed with cold PBS and lysed with Buffer A [10 mM Hepes/KOH pH 7.9, 1.5 mM MgCl2 , 10 mM KCl, 1 mM dithiothreitol (DTT), 0.2 mM EDTA, 0.1% Nonidet-P40, supplemented with a cocktail of protease (Roche Applied Science) and phosphatase inhibitors]. .. The cell suspension was then chilled on ice for 10 min before centrifugation at 10,000× g. The supernatant, corresponding to the cytosolic fraction, was then transferred to a fresh tube, while the resultant pellet was suspended in Buffer B [20 mM Hepes/KOH pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2 , 1 mM DTT, 0.2 mM EDTA, supplemented with the cocktail of protease (Roche Applied Science) and phosphatase inhibitors, 25 µg/ml leupeptin, 10 µg/ml pepstatin, 4 mM AEBSF and 100 µM MG-132] and incubated on ice for 20 min before being centrifuged at 10,000× g. Nuclear extract supernatant was collected, diluted 1∶4 in Buffer C [20 mM Hepes/KOH pH 7.9, 20% glycerol, 50 mM KCl, 1 mM DTT, 0.2 mM EDTA, 4 mM AEBSF, 25 µg/ml leupeptin, 5 µg/ml pepstatin, 100 µM MG-132] and stored in aliquots at −80°C until use.

Article Title: Affinity pulldown of γ-secretase and associated proteins from human and rat brain
Article Snippet: Cells were resuspended in 9 volumes of buffer A containing 20 mM Hepes, pH 7.5, 50 mM KCL, 2 mM EGTA and Complete™ protease inhibitor cocktail (which inhibits a broad spectrum of serine, cysteine and metalloproteases, Roche Applied Science, Indianapolis, IN, USA) and sonicated on ice for 30 sec. (Sonifier 450 BRANSON, Danbury, USA). .. Cell debris and nuclei were removed by centrifugation at 800 g for 10 min.

Article Title: Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis ▿
Article Snippet: Hen egg yolk sac-purified rickettsiae were concentrated to a final volume of 1 ml in modified buffer A (with Roche complete protease inhibitor cocktail at the manufacturer's suggested working concentration) and lysed by ballistic shearing using a Mini-Beadbeater apparatus (BioSpec Products), to deliver a 20-s pulse (maximum intensity setting), followed by incubation on ice for 2 min (repeated six times) using 0.1 mm zirconium beads. .. The lysate was cleared by centrifugation (10,000 × g , 4°C, 10 min), and the concentration of total protein was determined at an OD280 on a Nanodrop 2000 apparatus (Thermo Scientific).

Article Title: Characterization of the L-Lactate Dehydrogenase from Aggregatibacter actinomycetemcomitans
Article Snippet: Induced cells were harvested by centrifugation for 15 minutes at 6100×g in a Beckman Coulter Avanti J–E centrifuge at 4°C. .. The pellet was resuspended in 3 ml buffer A (25 mM phosphate buffer, 0.5 M NaCl, 20 mM imidazole, 10 µM flavin adenine dinucleotide (FAD), pH 7.07) containing one-half tablet complete Mini protease inhibitor cocktail (Roche), 25 U Benzonase Nuclease (Novagen) and 10 µM FAD (Alfa Aesar).

Filtration:

Article Title: Intramembrane Aromatic Interactions Influence the Lipid Sensitivities of Pentameric Ligand-gated Ion Channels *
Article Snippet: Cells were harvested, resuspended in Buffer A (150 m m NaCl, 50 m m NaH2 PO4 , pH 8.0) in the presence of either Roche CompleteTM antiprotease tablets (Branford, CT) or an analogous mix of locally prepared antiproteases and lysed with an Avestin Emulsiflex-C3 homogenizer (Ottawa, Canada). .. After treatment with herpes simplex 3C protease (Calbiochem), ELIC was eluted in 0.02% dodecylmaltoside and further purified in Buffer B (150 m m NaCl, 10 m m NaH2 PO4 , pH 8.0) on a Superose 6 10/300 gel filtration column (GE Healthcare).

Stable Transfection:

Article Title: Affinity pulldown of γ-secretase and associated proteins from human and rat brain
Article Snippet: γ-Secretase activity assay Blastocyst-derived ES-cells deficient in PS1 and PS2, stably expressing PS1, BD8-PS1 cells, were cultured as previously described [ ]. .. Cells were resuspended in 9 volumes of buffer A containing 20 mM Hepes, pH 7.5, 50 mM KCL, 2 mM EGTA and Complete™ protease inhibitor cocktail (which inhibits a broad spectrum of serine, cysteine and metalloproteases, Roche Applied Science, Indianapolis, IN, USA) and sonicated on ice for 30 sec. (Sonifier 450 BRANSON, Danbury, USA).

Synthesized:

Article Title: Exportin-5 mediates nuclear export of SRP RNA in vertebrates
Article Snippet: The competitor RNAs were also synthesized using MEGAscript. .. Biotinylated human SRP RNA was incubated with RanQ69LGTP and HeLa cell nuclear extracts, with or without the competitor RNAs in buffer A [20 mm Tris-HCl (pH7.5), 100 mm KCl, 2.5 mm MgCl2 , 20% glycerol, 0.5 mm DTT, 0.1% NP-40 and protease inhibitor cocktail, complete (Roche)] for 30 min at 30°C, and then the mixture was incubated with streptavidin-sepharose beads (GE Healthcare) for 1 h at 4°C.

Blocking Assay:

Article Title: Palytoxin and an Ostreopsis Toxin Extract Increase the Levels of mRNAs Encoding Inflammation-Related Proteins in Human Macrophages via p38 MAPK and NF-?B
Article Snippet: After treatment, cells were extensively washed with cold PBS and lysed with Buffer A [10 mM Hepes/KOH pH 7.9, 1.5 mM MgCl2 , 10 mM KCl, 1 mM dithiothreitol (DTT), 0.2 mM EDTA, 0.1% Nonidet-P40, supplemented with a cocktail of protease (Roche Applied Science) and phosphatase inhibitors]. .. To completely block in vitro degradation processes the buffer was further supplemented with the following protease inhibitors (Buffer A PLUS): 25 µg/ml leupeptin (Sigma Aldrich), 10 µg/ml pepstatin (Sigma Aldrich), 4 mM AEBSF (Roche Applied Science), 100 µM MG-132 (Enzo Life Sciences Inc., NY, USA).

Real-time Polymerase Chain Reaction:

Article Title: Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis ▿
Article Snippet: The reverse transcription reaction mixtures contained 500 ng of total R. prowazekii RNA, and the resulting cDNA was serially diluted to generate an internal standard curve for each qPCR, as described previously ( , ), using forward and reverse primers, each at a 128 nM final concentration. .. Hen egg yolk sac-purified rickettsiae were concentrated to a final volume of 1 ml in modified buffer A (with Roche complete protease inhibitor cocktail at the manufacturer's suggested working concentration) and lysed by ballistic shearing using a Mini-Beadbeater apparatus (BioSpec Products), to deliver a 20-s pulse (maximum intensity setting), followed by incubation on ice for 2 min (repeated six times) using 0.1 mm zirconium beads.

Incubation:

Article Title: Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases
Article Snippet: Immunoprecipitation (IP) IP was performed using buffer A (20 mM Tris-HCl, pH 7.2, 2 mM MgCl2 , 150 mM NaCl, 5 mM NaF, 1 mM Na3 VO4 , 0.5% NP-40, and protease inhibitor cocktail [Roche]). .. Cells were lysed in buffer A for 20 min on ice, followed by centrifugation at 13,000 g for 15 min. Primary antibodies (or anti-Flag M2-agarose affinity gel and anti-cdk5 (J3)–coupling gel [Pierce Chemical Co.]) were added to a final concentration of 5 μg/ml and incubated for 2 h to overnight at 4°C.

Article Title: Palytoxin and an Ostreopsis Toxin Extract Increase the Levels of mRNAs Encoding Inflammation-Related Proteins in Human Macrophages via p38 MAPK and NF-?B
Article Snippet: After treatment, cells were extensively washed with cold PBS and lysed with Buffer A [10 mM Hepes/KOH pH 7.9, 1.5 mM MgCl2 , 10 mM KCl, 1 mM dithiothreitol (DTT), 0.2 mM EDTA, 0.1% Nonidet-P40, supplemented with a cocktail of protease (Roche Applied Science) and phosphatase inhibitors]. .. The cell suspension was then chilled on ice for 10 min before centrifugation at 10,000× g. The supernatant, corresponding to the cytosolic fraction, was then transferred to a fresh tube, while the resultant pellet was suspended in Buffer B [20 mM Hepes/KOH pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2 , 1 mM DTT, 0.2 mM EDTA, supplemented with the cocktail of protease (Roche Applied Science) and phosphatase inhibitors, 25 µg/ml leupeptin, 10 µg/ml pepstatin, 4 mM AEBSF and 100 µM MG-132] and incubated on ice for 20 min before being centrifuged at 10,000× g. Nuclear extract supernatant was collected, diluted 1∶4 in Buffer C [20 mM Hepes/KOH pH 7.9, 20% glycerol, 50 mM KCl, 1 mM DTT, 0.2 mM EDTA, 4 mM AEBSF, 25 µg/ml leupeptin, 5 µg/ml pepstatin, 100 µM MG-132] and stored in aliquots at −80°C until use.

Article Title: Exportin-5 mediates nuclear export of SRP RNA in vertebrates
Article Snippet: .. Biotinylated human SRP RNA was incubated with RanQ69LGTP and HeLa cell nuclear extracts, with or without the competitor RNAs in buffer A [20 mm Tris-HCl (pH7.5), 100 mm KCl, 2.5 mm MgCl2 , 20% glycerol, 0.5 mm DTT, 0.1% NP-40 and protease inhibitor cocktail, complete (Roche)] for 30 min at 30°C, and then the mixture was incubated with streptavidin-sepharose beads (GE Healthcare) for 1 h at 4°C. .. After the beads were washed four times with buffer A, the bound material was recovered with 1 × SDS sample buffer and analyzed by Western blotting.

Article Title: Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis ▿
Article Snippet: .. Hen egg yolk sac-purified rickettsiae were concentrated to a final volume of 1 ml in modified buffer A (with Roche complete protease inhibitor cocktail at the manufacturer's suggested working concentration) and lysed by ballistic shearing using a Mini-Beadbeater apparatus (BioSpec Products), to deliver a 20-s pulse (maximum intensity setting), followed by incubation on ice for 2 min (repeated six times) using 0.1 mm zirconium beads. .. The lysate was cleared by centrifugation (10,000 × g , 4°C, 10 min), and the concentration of total protein was determined at an OD280 on a Nanodrop 2000 apparatus (Thermo Scientific).

Article Title: Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation
Article Snippet: The cell pellet was resuspended in 5 times the volume of Buffer A (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and Complete Protease Inhibitor; Roche) supplemented with 250 mM sucrose. .. The resuspended cell pellet was incubated on ice for 15 min before the cells were broken by passing them through a 22-gauge needle 25 times.

Activity Assay:

Article Title: Affinity pulldown of γ-secretase and associated proteins from human and rat brain
Article Snippet: Paragraph title: γ-Secretase activity assay ... Cells were resuspended in 9 volumes of buffer A containing 20 mM Hepes, pH 7.5, 50 mM KCL, 2 mM EGTA and Complete™ protease inhibitor cocktail (which inhibits a broad spectrum of serine, cysteine and metalloproteases, Roche Applied Science, Indianapolis, IN, USA) and sonicated on ice for 30 sec. (Sonifier 450 BRANSON, Danbury, USA).

Article Title: Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis ▿
Article Snippet: Paragraph title: gpsA mRNA detection by qRT-PCR and detection of G3PDH activity in R. prowazekii lysed-cell extracts. ... Hen egg yolk sac-purified rickettsiae were concentrated to a final volume of 1 ml in modified buffer A (with Roche complete protease inhibitor cocktail at the manufacturer's suggested working concentration) and lysed by ballistic shearing using a Mini-Beadbeater apparatus (BioSpec Products), to deliver a 20-s pulse (maximum intensity setting), followed by incubation on ice for 2 min (repeated six times) using 0.1 mm zirconium beads.

Expressing:

Article Title: The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding
Article Snippet: Expression of the plasmid-encoded genes was achieved by induction with 1 mM IPTG (30 min), followed by addition of rifampicin (200 µg/ml) and growth for an additional 90 min. .. The cell pellet was thawed and resuspended (100× concentrated) in buffer A [20 mM Tris–HCl pH 7.6, 1 mM EDTA, 1 mM dithiothreitol, 5% (v/v) glycerol] plus 1 M NaCl and a tablet of protease inhibitor cocktail (Complete, Roche).

Article Title: Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles
Article Snippet: .. Briefly, protein expression was induced in the presence of 0.5 mM IPTG and 10 μM FAD at 16°C for 16–20 h. The induced cell pellets were resuspended in buffer A [150 mM NaCl and 50 mM Tris/HCl (pH 7.4)] supplemented with 5 mM imidazole, 50 μM FAD and one tablet of EDTA-free protease inhibitor cocktail (Roche) and then sonicated on ice. ..

Article Title: Affinity pulldown of γ-secretase and associated proteins from human and rat brain
Article Snippet: γ-Secretase activity assay Blastocyst-derived ES-cells deficient in PS1 and PS2, stably expressing PS1, BD8-PS1 cells, were cultured as previously described [ ]. .. Cells were resuspended in 9 volumes of buffer A containing 20 mM Hepes, pH 7.5, 50 mM KCL, 2 mM EGTA and Complete™ protease inhibitor cocktail (which inhibits a broad spectrum of serine, cysteine and metalloproteases, Roche Applied Science, Indianapolis, IN, USA) and sonicated on ice for 30 sec. (Sonifier 450 BRANSON, Danbury, USA).

Cell Fractionation:

Article Title: Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation
Article Snippet: Paragraph title: UV treatment and cellular fractionation ... The cell pellet was resuspended in 5 times the volume of Buffer A (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and Complete Protease Inhibitor; Roche) supplemented with 250 mM sucrose.

Modification:

Article Title: Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis ▿
Article Snippet: .. Hen egg yolk sac-purified rickettsiae were concentrated to a final volume of 1 ml in modified buffer A (with Roche complete protease inhibitor cocktail at the manufacturer's suggested working concentration) and lysed by ballistic shearing using a Mini-Beadbeater apparatus (BioSpec Products), to deliver a 20-s pulse (maximum intensity setting), followed by incubation on ice for 2 min (repeated six times) using 0.1 mm zirconium beads. .. The lysate was cleared by centrifugation (10,000 × g , 4°C, 10 min), and the concentration of total protein was determined at an OD280 on a Nanodrop 2000 apparatus (Thermo Scientific).

Western Blot:

Article Title: Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases
Article Snippet: Immunoprecipitation (IP) IP was performed using buffer A (20 mM Tris-HCl, pH 7.2, 2 mM MgCl2 , 150 mM NaCl, 5 mM NaF, 1 mM Na3 VO4 , 0.5% NP-40, and protease inhibitor cocktail [Roche]). .. Anti–mouse or –rabbit IgG agarose were added to the mixture and incubated at 4°C for 1 h. After three washings, IP products were either directly boiled in Laemmli buffer or eluted with 0.1 M glycine, pH 2.3, and then boiled in Laemmli buffer and subjected to PVDF membrane transfer and Western blot.

Article Title: Exportin-5 mediates nuclear export of SRP RNA in vertebrates
Article Snippet: Biotinylated human SRP RNA was incubated with RanQ69LGTP and HeLa cell nuclear extracts, with or without the competitor RNAs in buffer A [20 mm Tris-HCl (pH7.5), 100 mm KCl, 2.5 mm MgCl2 , 20% glycerol, 0.5 mm DTT, 0.1% NP-40 and protease inhibitor cocktail, complete (Roche)] for 30 min at 30°C, and then the mixture was incubated with streptavidin-sepharose beads (GE Healthcare) for 1 h at 4°C. .. After the beads were washed four times with buffer A, the bound material was recovered with 1 × SDS sample buffer and analyzed by Western blotting.

Transformation Assay:

Article Title: Intramembrane Aromatic Interactions Influence the Lipid Sensitivities of Pentameric Ligand-gated Ion Channels *
Article Snippet: Cultures of transformed cells were grown in Terrific Broth containing 50 μg/ml kanamycin at 37 °C to an A 600 of ∼1.2 absorbance units and then induced overnight at 26 °C with 200 μ m isopropyl 1-thio-β- d -galactopyranoside. .. Cells were harvested, resuspended in Buffer A (150 m m NaCl, 50 m m NaH2 PO4 , pH 8.0) in the presence of either Roche CompleteTM antiprotease tablets (Branford, CT) or an analogous mix of locally prepared antiproteases and lysed with an Avestin Emulsiflex-C3 homogenizer (Ottawa, Canada).

Immunoprecipitation:

Article Title: Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases
Article Snippet: .. Immunoprecipitation (IP) IP was performed using buffer A (20 mM Tris-HCl, pH 7.2, 2 mM MgCl2 , 150 mM NaCl, 5 mM NaF, 1 mM Na3 VO4 , 0.5% NP-40, and protease inhibitor cocktail [Roche]). .. Cells were lysed in buffer A for 20 min on ice, followed by centrifugation at 13,000 g for 15 min. Primary antibodies (or anti-Flag M2-agarose affinity gel and anti-cdk5 (J3)–coupling gel [Pierce Chemical Co.]) were added to a final concentration of 5 μg/ml and incubated for 2 h to overnight at 4°C.

Protease Inhibitor:

Article Title: The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban
Article Snippet: .. Testes from two mice were homogenised in cold 1.5 ml buffer A (250 mM sucrose, 50 mM HEPES/KOH, pH 7.6, 50 mM potassium acetate, 6 mM magnesium acetate, 1 mM EDTA, 1 mM DTT) containing 10 μg/ml PMSF and EDTA‐free complete protease inhibitor cocktail (Roche) using a glass–Teflon potter at full speed. .. The supernatant was collected and kept on ice, and the pellet was resuspended in 1.5 ml buffer A followed by homogenisation and centrifugation as described above.

Article Title: Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases
Article Snippet: .. Immunoprecipitation (IP) IP was performed using buffer A (20 mM Tris-HCl, pH 7.2, 2 mM MgCl2 , 150 mM NaCl, 5 mM NaF, 1 mM Na3 VO4 , 0.5% NP-40, and protease inhibitor cocktail [Roche]). .. Cells were lysed in buffer A for 20 min on ice, followed by centrifugation at 13,000 g for 15 min. Primary antibodies (or anti-Flag M2-agarose affinity gel and anti-cdk5 (J3)–coupling gel [Pierce Chemical Co.]) were added to a final concentration of 5 μg/ml and incubated for 2 h to overnight at 4°C.

Article Title: The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding
Article Snippet: .. The cell pellet was thawed and resuspended (100× concentrated) in buffer A [20 mM Tris–HCl pH 7.6, 1 mM EDTA, 1 mM dithiothreitol, 5% (v/v) glycerol] plus 1 M NaCl and a tablet of protease inhibitor cocktail (Complete, Roche). ..

Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis
Article Snippet: .. Subcellular fractionation Cells were resuspended in hypotonic buffer A (250 mM sucrose, 20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 1× protease inhibitor complex [Roche]) on ice for 30 min. ..

Article Title: Exportin-5 mediates nuclear export of SRP RNA in vertebrates
Article Snippet: .. Biotinylated human SRP RNA was incubated with RanQ69LGTP and HeLa cell nuclear extracts, with or without the competitor RNAs in buffer A [20 mm Tris-HCl (pH7.5), 100 mm KCl, 2.5 mm MgCl2 , 20% glycerol, 0.5 mm DTT, 0.1% NP-40 and protease inhibitor cocktail, complete (Roche)] for 30 min at 30°C, and then the mixture was incubated with streptavidin-sepharose beads (GE Healthcare) for 1 h at 4°C. .. After the beads were washed four times with buffer A, the bound material was recovered with 1 × SDS sample buffer and analyzed by Western blotting.

Article Title: Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles
Article Snippet: .. Briefly, protein expression was induced in the presence of 0.5 mM IPTG and 10 μM FAD at 16°C for 16–20 h. The induced cell pellets were resuspended in buffer A [150 mM NaCl and 50 mM Tris/HCl (pH 7.4)] supplemented with 5 mM imidazole, 50 μM FAD and one tablet of EDTA-free protease inhibitor cocktail (Roche) and then sonicated on ice. ..

Article Title: Affinity pulldown of γ-secretase and associated proteins from human and rat brain
Article Snippet: .. Cells were resuspended in 9 volumes of buffer A containing 20 mM Hepes, pH 7.5, 50 mM KCL, 2 mM EGTA and Complete™ protease inhibitor cocktail (which inhibits a broad spectrum of serine, cysteine and metalloproteases, Roche Applied Science, Indianapolis, IN, USA) and sonicated on ice for 30 sec. (Sonifier 450 BRANSON, Danbury, USA). .. Cell debris and nuclei were removed by centrifugation at 800 g for 10 min.

Article Title: Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis ▿
Article Snippet: .. Hen egg yolk sac-purified rickettsiae were concentrated to a final volume of 1 ml in modified buffer A (with Roche complete protease inhibitor cocktail at the manufacturer's suggested working concentration) and lysed by ballistic shearing using a Mini-Beadbeater apparatus (BioSpec Products), to deliver a 20-s pulse (maximum intensity setting), followed by incubation on ice for 2 min (repeated six times) using 0.1 mm zirconium beads. .. The lysate was cleared by centrifugation (10,000 × g , 4°C, 10 min), and the concentration of total protein was determined at an OD280 on a Nanodrop 2000 apparatus (Thermo Scientific).

Article Title: Characterization of the L-Lactate Dehydrogenase from Aggregatibacter actinomycetemcomitans
Article Snippet: .. The pellet was resuspended in 3 ml buffer A (25 mM phosphate buffer, 0.5 M NaCl, 20 mM imidazole, 10 µM flavin adenine dinucleotide (FAD), pH 7.07) containing one-half tablet complete Mini protease inhibitor cocktail (Roche), 25 U Benzonase Nuclease (Novagen) and 10 µM FAD (Alfa Aesar). ..

Article Title: Complicated behavior of G-quadruplexes and evaluating G-quadruplexes' ligands in various systems mimicking cellular circumstance
Article Snippet: .. Frozen cells were quickly thawed in a water bath at 37 °C and washed with 5 packed-cell volume (pcv) of ice-cold Buffer A, which contained 20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, and 1 tablet of protease inhibitor (Roche complete, Mini, EDTA-free protease inhibitor cocktail tablets) for 10 mL of Buffer A. ..

Article Title: Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation
Article Snippet: .. The cell pellet was resuspended in 5 times the volume of Buffer A (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and Complete Protease Inhibitor; Roche) supplemented with 250 mM sucrose. .. The resuspended cell pellet was incubated on ice for 15 min before the cells were broken by passing them through a 22-gauge needle 25 times.

Cell Culture:

Article Title: A novel Apaf-1-independent putative caspase-2 activation complex
Article Snippet: Paragraph title: Cell culture and preparation of lysates ... To prepare lysates, cells were washed twice with PBS and resuspended in buffer A (20 mM Hepes-KOH, 10 mM KCl, 1 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, pH 7.5) supplemented with protease inhibitors (Complete™; Roche).

Article Title: Affinity pulldown of γ-secretase and associated proteins from human and rat brain
Article Snippet: γ-Secretase activity assay Blastocyst-derived ES-cells deficient in PS1 and PS2, stably expressing PS1, BD8-PS1 cells, were cultured as previously described [ ]. .. Cells were resuspended in 9 volumes of buffer A containing 20 mM Hepes, pH 7.5, 50 mM KCL, 2 mM EGTA and Complete™ protease inhibitor cocktail (which inhibits a broad spectrum of serine, cysteine and metalloproteases, Roche Applied Science, Indianapolis, IN, USA) and sonicated on ice for 30 sec. (Sonifier 450 BRANSON, Danbury, USA).

Article Title: Complicated behavior of G-quadruplexes and evaluating G-quadruplexes' ligands in various systems mimicking cellular circumstance
Article Snippet: The cell culture was maintained in an RPMI-1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified atmosphere with 5% CO2 . .. Frozen cells were quickly thawed in a water bath at 37 °C and washed with 5 packed-cell volume (pcv) of ice-cold Buffer A, which contained 20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, and 1 tablet of protease inhibitor (Roche complete, Mini, EDTA-free protease inhibitor cocktail tablets) for 10 mL of Buffer A.

Sequencing:

Article Title: The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding
Article Snippet: Paragraph title: Protein purification and N-terminal sequencing ... The cell pellet was thawed and resuspended (100× concentrated) in buffer A [20 mM Tris–HCl pH 7.6, 1 mM EDTA, 1 mM dithiothreitol, 5% (v/v) glycerol] plus 1 M NaCl and a tablet of protease inhibitor cocktail (Complete, Roche).

Sonication:

Article Title: Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles
Article Snippet: .. Briefly, protein expression was induced in the presence of 0.5 mM IPTG and 10 μM FAD at 16°C for 16–20 h. The induced cell pellets were resuspended in buffer A [150 mM NaCl and 50 mM Tris/HCl (pH 7.4)] supplemented with 5 mM imidazole, 50 μM FAD and one tablet of EDTA-free protease inhibitor cocktail (Roche) and then sonicated on ice. ..

Article Title: Affinity pulldown of γ-secretase and associated proteins from human and rat brain
Article Snippet: .. Cells were resuspended in 9 volumes of buffer A containing 20 mM Hepes, pH 7.5, 50 mM KCL, 2 mM EGTA and Complete™ protease inhibitor cocktail (which inhibits a broad spectrum of serine, cysteine and metalloproteases, Roche Applied Science, Indianapolis, IN, USA) and sonicated on ice for 30 sec. (Sonifier 450 BRANSON, Danbury, USA). .. Cell debris and nuclei were removed by centrifugation at 800 g for 10 min.

Affinity Purification:

Article Title: ABIN-2 Forms a Ternary Complex with TPL-2 and NF-?B1 p105 and Is Essential for TPL-2 Protein Stability †
Article Snippet: Paragraph title: Affinity purification of HA-p105(S927A). ... Cells were lysed in 10 volumes of ice-cold buffer A (1% Nonidet P-40 [NP-40], 50 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 1 mM Na3 VO4 , 1 mM Na4 P2 O7 plus a mixture of protease inhibitors [Roche Molecular Biochemicals]).

Binding Assay:

Article Title: ABIN-2 Forms a Ternary Complex with TPL-2 and NF-?B1 p105 and Is Essential for TPL-2 Protein Stability †
Article Snippet: Cells were lysed in 10 volumes of ice-cold buffer A (1% Nonidet P-40 [NP-40], 50 mM Tris [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 1 mM Na3 VO4 , 1 mM Na4 P2 O7 plus a mixture of protease inhibitors [Roche Molecular Biochemicals]). .. Lysates were precleared of nonspecific binding proteins first by two sequential batch incubations (overnight and 2 h) with 1-ml aliquots of protein A-Sepharose beads (Amersham Biosciences).

Article Title: Exportin-5 mediates nuclear export of SRP RNA in vertebrates
Article Snippet: Paragraph title: RNA-protein binding assay ... Biotinylated human SRP RNA was incubated with RanQ69LGTP and HeLa cell nuclear extracts, with or without the competitor RNAs in buffer A [20 mm Tris-HCl (pH7.5), 100 mm KCl, 2.5 mm MgCl2 , 20% glycerol, 0.5 mm DTT, 0.1% NP-40 and protease inhibitor cocktail, complete (Roche)] for 30 min at 30°C, and then the mixture was incubated with streptavidin-sepharose beads (GE Healthcare) for 1 h at 4°C.

Article Title: Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles
Article Snippet: Briefly, protein expression was induced in the presence of 0.5 mM IPTG and 10 μM FAD at 16°C for 16–20 h. The induced cell pellets were resuspended in buffer A [150 mM NaCl and 50 mM Tris/HCl (pH 7.4)] supplemented with 5 mM imidazole, 50 μM FAD and one tablet of EDTA-free protease inhibitor cocktail (Roche) and then sonicated on ice. .. Bind resin (Novagen) pre-equilibrated with binding buffer.

Pull Down Assay:

Article Title: Exportin-5 mediates nuclear export of SRP RNA in vertebrates
Article Snippet: Biotinylated human SRP RNA was incubated with RanQ69LGTP and HeLa cell nuclear extracts, with or without the competitor RNAs in buffer A [20 mm Tris-HCl (pH7.5), 100 mm KCl, 2.5 mm MgCl2 , 20% glycerol, 0.5 mm DTT, 0.1% NP-40 and protease inhibitor cocktail, complete (Roche)] for 30 min at 30°C, and then the mixture was incubated with streptavidin-sepharose beads (GE Healthcare) for 1 h at 4°C. .. The pull-down assay using the recombinant Exportin-5 was similarly carried out.

Isolation:

Article Title: The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban
Article Snippet: Paragraph title: Microsome isolation from mouse testis and blue‐native (BN) PAGE ... Testes from two mice were homogenised in cold 1.5 ml buffer A (250 mM sucrose, 50 mM HEPES/KOH, pH 7.6, 50 mM potassium acetate, 6 mM magnesium acetate, 1 mM EDTA, 1 mM DTT) containing 10 μg/ml PMSF and EDTA‐free complete protease inhibitor cocktail (Roche) using a glass–Teflon potter at full speed.

Size-exclusion Chromatography:

Article Title: Affinity pulldown of γ-secretase and associated proteins from human and rat brain
Article Snippet: .. Cells were resuspended in 9 volumes of buffer A containing 20 mM Hepes, pH 7.5, 50 mM KCL, 2 mM EGTA and Complete™ protease inhibitor cocktail (which inhibits a broad spectrum of serine, cysteine and metalloproteases, Roche Applied Science, Indianapolis, IN, USA) and sonicated on ice for 30 sec. (Sonifier 450 BRANSON, Danbury, USA). .. Cell debris and nuclei were removed by centrifugation at 800 g for 10 min.

Purification:

Article Title: The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding
Article Snippet: Protein purification and N-terminal sequencing MobM and MobMN199 proteins were overproduced and purified by a newly developed protocol, with greater speed and higher yield than the one previously described for MobM ( , ). .. The cell pellet was thawed and resuspended (100× concentrated) in buffer A [20 mM Tris–HCl pH 7.6, 1 mM EDTA, 1 mM dithiothreitol, 5% (v/v) glycerol] plus 1 M NaCl and a tablet of protease inhibitor cocktail (Complete, Roche).

Article Title: Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis ▿
Article Snippet: Purification of total R. prowazekii RNA and quantitative reverse transcriptase (qRT) PCR (qRT-PCR) protocols were performed as described previously ( , ) using the Express Two-Step qRT-PCR system from Invitrogen, according to the manufacturer's directions. .. Hen egg yolk sac-purified rickettsiae were concentrated to a final volume of 1 ml in modified buffer A (with Roche complete protease inhibitor cocktail at the manufacturer's suggested working concentration) and lysed by ballistic shearing using a Mini-Beadbeater apparatus (BioSpec Products), to deliver a 20-s pulse (maximum intensity setting), followed by incubation on ice for 2 min (repeated six times) using 0.1 mm zirconium beads.

Article Title: Intramembrane Aromatic Interactions Influence the Lipid Sensitivities of Pentameric Ligand-gated Ion Channels *
Article Snippet: ELIC was expressed, purified, and reconstituted into proteoliposomes as described previously for GLIC ( ) but with several modifications. .. Cells were harvested, resuspended in Buffer A (150 m m NaCl, 50 m m NaH2 PO4 , pH 8.0) in the presence of either Roche CompleteTM antiprotease tablets (Branford, CT) or an analogous mix of locally prepared antiproteases and lysed with an Avestin Emulsiflex-C3 homogenizer (Ottawa, Canada).

Protein Purification:

Article Title: The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding
Article Snippet: Paragraph title: Protein purification and N-terminal sequencing ... The cell pellet was thawed and resuspended (100× concentrated) in buffer A [20 mM Tris–HCl pH 7.6, 1 mM EDTA, 1 mM dithiothreitol, 5% (v/v) glycerol] plus 1 M NaCl and a tablet of protease inhibitor cocktail (Complete, Roche).

Article Title: Mitochondrial thiol oxidase Erv1: both shuttle cysteine residues are required for its function with distinct roles
Article Snippet: Paragraph title: Protein purification ... Briefly, protein expression was induced in the presence of 0.5 mM IPTG and 10 μM FAD at 16°C for 16–20 h. The induced cell pellets were resuspended in buffer A [150 mM NaCl and 50 mM Tris/HCl (pH 7.4)] supplemented with 5 mM imidazole, 50 μM FAD and one tablet of EDTA-free protease inhibitor cocktail (Roche) and then sonicated on ice.

Article Title: Characterization of the L-Lactate Dehydrogenase from Aggregatibacter actinomycetemcomitans
Article Snippet: Paragraph title: Protein Purification ... The pellet was resuspended in 3 ml buffer A (25 mM phosphate buffer, 0.5 M NaCl, 20 mM imidazole, 10 µM flavin adenine dinucleotide (FAD), pH 7.07) containing one-half tablet complete Mini protease inhibitor cocktail (Roche), 25 U Benzonase Nuclease (Novagen) and 10 µM FAD (Alfa Aesar).

Quantitative RT-PCR:

Article Title: Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis ▿
Article Snippet: Paragraph title: gpsA mRNA detection by qRT-PCR and detection of G3PDH activity in R. prowazekii lysed-cell extracts. ... Hen egg yolk sac-purified rickettsiae were concentrated to a final volume of 1 ml in modified buffer A (with Roche complete protease inhibitor cocktail at the manufacturer's suggested working concentration) and lysed by ballistic shearing using a Mini-Beadbeater apparatus (BioSpec Products), to deliver a 20-s pulse (maximum intensity setting), followed by incubation on ice for 2 min (repeated six times) using 0.1 mm zirconium beads.

Polyacrylamide Gel Electrophoresis:

Article Title: The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban
Article Snippet: Paragraph title: Microsome isolation from mouse testis and blue‐native (BN) PAGE ... Testes from two mice were homogenised in cold 1.5 ml buffer A (250 mM sucrose, 50 mM HEPES/KOH, pH 7.6, 50 mM potassium acetate, 6 mM magnesium acetate, 1 mM EDTA, 1 mM DTT) containing 10 μg/ml PMSF and EDTA‐free complete protease inhibitor cocktail (Roche) using a glass–Teflon potter at full speed.

Mouse Assay:

Article Title: The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban
Article Snippet: .. Testes from two mice were homogenised in cold 1.5 ml buffer A (250 mM sucrose, 50 mM HEPES/KOH, pH 7.6, 50 mM potassium acetate, 6 mM magnesium acetate, 1 mM EDTA, 1 mM DTT) containing 10 μg/ml PMSF and EDTA‐free complete protease inhibitor cocktail (Roche) using a glass–Teflon potter at full speed. .. The supernatant was collected and kept on ice, and the pellet was resuspended in 1.5 ml buffer A followed by homogenisation and centrifugation as described above.

Plasmid Preparation:

Article Title: The MobM relaxase domain of plasmid pMV158: thermal stability and activity upon Mn2+ and specific DNA binding
Article Snippet: Expression of the plasmid-encoded genes was achieved by induction with 1 mM IPTG (30 min), followed by addition of rifampicin (200 µg/ml) and growth for an additional 90 min. .. The cell pellet was thawed and resuspended (100× concentrated) in buffer A [20 mM Tris–HCl pH 7.6, 1 mM EDTA, 1 mM dithiothreitol, 5% (v/v) glycerol] plus 1 M NaCl and a tablet of protease inhibitor cocktail (Complete, Roche).

Irradiation:

Article Title: Elimination of Mcl-1 is required for the initiation of apoptosis following ultraviolet irradiation
Article Snippet: The cover of each dish was removed before the cells were treated in a Stratagene stratalinker with 200 mJ/cm2 of UV irradiation (254 nm). .. The cell pellet was resuspended in 5 times the volume of Buffer A (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM PMSF, and Complete Protease Inhibitor; Roche) supplemented with 250 mM sucrose.

Recombinant:

Article Title: A novel Apaf-1-independent putative caspase-2 activation complex
Article Snippet: Apaf-1 wild-type and Apaf-1−/− cells (retrovirally immortalized murine fetal liver cells) were maintained in DME with 10% FBS, 1 mM glutamine and 1/4,000 recombinant murine IL-3 (a gift from A.F. .. To prepare lysates, cells were washed twice with PBS and resuspended in buffer A (20 mM Hepes-KOH, 10 mM KCl, 1 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 1 mM DTT, pH 7.5) supplemented with protease inhibitors (Complete™; Roche).

Article Title: Exportin-5 mediates nuclear export of SRP RNA in vertebrates
Article Snippet: Biotinylated human SRP RNA was incubated with RanQ69LGTP and HeLa cell nuclear extracts, with or without the competitor RNAs in buffer A [20 mm Tris-HCl (pH7.5), 100 mm KCl, 2.5 mm MgCl2 , 20% glycerol, 0.5 mm DTT, 0.1% NP-40 and protease inhibitor cocktail, complete (Roche)] for 30 min at 30°C, and then the mixture was incubated with streptavidin-sepharose beads (GE Healthcare) for 1 h at 4°C. .. The pull-down assay using the recombinant Exportin-5 was similarly carried out.

Article Title: Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis ▿
Article Snippet: Hen egg yolk sac-purified rickettsiae were concentrated to a final volume of 1 ml in modified buffer A (with Roche complete protease inhibitor cocktail at the manufacturer's suggested working concentration) and lysed by ballistic shearing using a Mini-Beadbeater apparatus (BioSpec Products), to deliver a 20-s pulse (maximum intensity setting), followed by incubation on ice for 2 min (repeated six times) using 0.1 mm zirconium beads. .. G3PDH assays were carried out as described above for the purified recombinant N-His6 -GpsA protein with the rickettsial lysed-cell extract and substrate concentrations reported in the legend to Fig. .

In Vitro:

Article Title: Palytoxin and an Ostreopsis Toxin Extract Increase the Levels of mRNAs Encoding Inflammation-Related Proteins in Human Macrophages via p38 MAPK and NF-?B
Article Snippet: After treatment, cells were extensively washed with cold PBS and lysed with Buffer A [10 mM Hepes/KOH pH 7.9, 1.5 mM MgCl2 , 10 mM KCl, 1 mM dithiothreitol (DTT), 0.2 mM EDTA, 0.1% Nonidet-P40, supplemented with a cocktail of protease (Roche Applied Science) and phosphatase inhibitors]. .. To completely block in vitro degradation processes the buffer was further supplemented with the following protease inhibitors (Buffer A PLUS): 25 µg/ml leupeptin (Sigma Aldrich), 10 µg/ml pepstatin (Sigma Aldrich), 4 mM AEBSF (Roche Applied Science), 100 µM MG-132 (Enzo Life Sciences Inc., NY, USA).

Homogenization:

Article Title: The intramembrane protease SPPL2c promotes male germ cell development by cleaving phospholamban
Article Snippet: Testes from two mice were homogenised in cold 1.5 ml buffer A (250 mM sucrose, 50 mM HEPES/KOH, pH 7.6, 50 mM potassium acetate, 6 mM magnesium acetate, 1 mM EDTA, 1 mM DTT) containing 10 μg/ml PMSF and EDTA‐free complete protease inhibitor cocktail (Roche) using a glass–Teflon potter at full speed. .. The supernatant was collected and kept on ice, and the pellet was resuspended in 1.5 ml buffer A followed by homogenisation and centrifugation as described above.

Concentration Assay:

Article Title: Cdk5 phosphorylation of huntingtin reduces its cleavage by caspases
Article Snippet: Immunoprecipitation (IP) IP was performed using buffer A (20 mM Tris-HCl, pH 7.2, 2 mM MgCl2 , 150 mM NaCl, 5 mM NaF, 1 mM Na3 VO4 , 0.5% NP-40, and protease inhibitor cocktail [Roche]). .. Cells were lysed in buffer A for 20 min on ice, followed by centrifugation at 13,000 g for 15 min. Primary antibodies (or anti-Flag M2-agarose affinity gel and anti-cdk5 (J3)–coupling gel [Pierce Chemical Co.]) were added to a final concentration of 5 μg/ml and incubated for 2 h to overnight at 4°C.

Article Title: Rickettsia prowazekii Uses an sn-Glycerol-3-Phosphate Dehydrogenase and a Novel Dihydroxyacetone Phosphate Transport System To Supply Triose Phosphate for Phospholipid Biosynthesis ▿
Article Snippet: .. Hen egg yolk sac-purified rickettsiae were concentrated to a final volume of 1 ml in modified buffer A (with Roche complete protease inhibitor cocktail at the manufacturer's suggested working concentration) and lysed by ballistic shearing using a Mini-Beadbeater apparatus (BioSpec Products), to deliver a 20-s pulse (maximum intensity setting), followed by incubation on ice for 2 min (repeated six times) using 0.1 mm zirconium beads. .. The lysate was cleared by centrifugation (10,000 × g , 4°C, 10 min), and the concentration of total protein was determined at an OD280 on a Nanodrop 2000 apparatus (Thermo Scientific).

Fractionation:

Article Title: Bax and Bak can localize to the endoplasmic reticulum to initiate apoptosis
Article Snippet: .. Subcellular fractionation Cells were resuspended in hypotonic buffer A (250 mM sucrose, 20 mM Hepes, pH 7.5, 10 mM KCl, 1.5 mM MgCl2 , 1 mM EDTA, 1 mM EGTA, 1× protease inhibitor complex [Roche]) on ice for 30 min. ..

Article Title: Palytoxin and an Ostreopsis Toxin Extract Increase the Levels of mRNAs Encoding Inflammation-Related Proteins in Human Macrophages via p38 MAPK and NF-?B
Article Snippet: Paragraph title: Nuclear-cytoplasmic subcellular fractionation ... After treatment, cells were extensively washed with cold PBS and lysed with Buffer A [10 mM Hepes/KOH pH 7.9, 1.5 mM MgCl2 , 10 mM KCl, 1 mM dithiothreitol (DTT), 0.2 mM EDTA, 0.1% Nonidet-P40, supplemented with a cocktail of protease (Roche Applied Science) and phosphatase inhibitors].

Lysis:

Article Title: Palytoxin and an Ostreopsis Toxin Extract Increase the Levels of mRNAs Encoding Inflammation-Related Proteins in Human Macrophages via p38 MAPK and NF-?B
Article Snippet: Nuclear-cytoplasmic subcellular fractionation Cytosolic and nuclear extracts were obtained by low salt/detergent cell lysis followed by high salt extraction of nuclei as previously described . .. After treatment, cells were extensively washed with cold PBS and lysed with Buffer A [10 mM Hepes/KOH pH 7.9, 1.5 mM MgCl2 , 10 mM KCl, 1 mM dithiothreitol (DTT), 0.2 mM EDTA, 0.1% Nonidet-P40, supplemented with a cocktail of protease (Roche Applied Science) and phosphatase inhibitors].

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    Roche immunoprecipitation buffer
    CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of <t>co-immunoprecipitation</t> experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).
    Immunoprecipitation Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 228 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 228 article reviews
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    99
    Roche flag lysis buffer
    In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with <t>anti-FLAG</t> antibodies, resolved by SDS-PAGE, and detected by western blotting using the <t>M2</t> anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.
    Flag Lysis Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flag lysis buffer/product/Roche
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    99
    Roche nonidet p 40 buffer
    Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery. A , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting ( IB ) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8.  Asterisk  denotes the heavy chain of the antibody used for IP.  Solid  and  open arrowheads  indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments.  B , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 ( WT ) or the K273A TRAIL-R1 mutant ( K / A ), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments.  C , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for  panel A. Asterisk  denotes the heavy chain of the antibody used for IP.  Solid  and  open arrowheads  indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments.  D , alignment of primary amino acid sequence of part of the transmembrane segment ( italic ) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in  E . Relevant potential ubiquitination sites are shown in  bold. E , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in  D . TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments.  Asterisk  denotes the heavy chain of the antibody used for IP.
    Nonidet P 40 Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Roche tris buffer
    Localization of TcdE in E. coli and C. difficile . A. Cytoplasmic and membrane proteins analysis of E. coli lysogens of λCmrΔ(SR) carrying pBR322 (control) or pCD463 (+ tcdE -6xHis). B. Cytoplasmic and membrane proteins analysis of C. difficile strain carrying either pMTL84151 (control) or pRG46 (+ tcdE -6xHis). (1) SDS-PAGE coomassie stained gel. Western blots probed with 6XHis Tag antibody (2), ATPase Beta subunit antibody (3), and Ribosomal subunits LI/L2 monoclonal antibody (4). C. Membrane protein samples from bacterial cells expressing TcdE-6His <t>resuspended</t> in denature or native sample buffers and analyzed by Western blot using His-Tag antibody. D. Membrane proteins of JIR8094, TcdE mutant and complemented TcdE mutant strains were harvested from bacterial cultures induced with 20 ng/ml of ATc for 2 hours, separated in 16% <t>Tris-Glycine</t> gel and transferred into PVDF membrane. Panels 1. Ponceau stained membrane; 2. Probed with TcdE antibody.
    Tris Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

    Journal: Molecular psychiatry

    Article Title: CNTNAP2 stabilizes interneuron dendritic arbors through CASK

    doi: 10.1038/s41380-018-0027-3

    Figure Lengend Snippet: CNTNAP2 interacts with CASK at the plasma membrane in cortical GABAergic interneurons (a) Only yeast cells co-expressing CNTNAP2 bait and CASK prey constructs grow on high stringency yeast plates (QDO/X/A). (b–c) Cropped western blots of co-immunoprecipitation experiments with CASK and CNTNAP2 in mouse cortex. (d) Cropped western blots showing co-immunoprecipitation of various FLAG-CNTNAP2 truncation mutants ( Supplementary Figure 5a ; red lines) with untagged, full-length CASK in HEK293T cells. (e) Representative cropped western blots of membrane/cytosol fractions of HEK293T cells expressing pCS2-FLAG + CASK-mCherry (CASK), FLAG-CNTNAP2 + CASK-mCherry (CNTNAP2 + CASK), or FLAG-CNTNAP2 + CASKΔPDZ-mCherry (CNTNAP2 + CASKΔPDZ) and subsequent quantification of protein localization (CASK vs. CNTNAP2 + CASK vs. CNTNAP2 + CASKΔPDZ: 6 independent experiments; CASK alone vs. CASKΔPDZ alone: 3 independent experiments). Percentages were calculated by dividing the densitometry value of CASK/CNTNAP2 in either membrane or cytosol fraction by the summation of both. (f) Representative SIM image of endogenous CNTNAP2 and CASK co-localization (white) on a GFP-transfected interneuronal dendrite (scale bar = 5 μm). (g) Histogram showing distribution of CASK/CNTNAP2 co-localized puncta relative to the dendrite’s lateral edge from (f) (CASK colocalized with CNTNAP2: n = 79 puncta from 3 cultures; CNTNAP2 colocalized with CASK: n = 70 puncta from 3 cultures). (h) Representative confocal image showing PLA signal from endogenous CASK/CNTNAP2 staining, which occurs only when CASK and CNTNAP2 primary antibodies are both applied (scale bar = 1 μm). (i) Cropped immunoblots of subcellular fractionations from adult mouse forebrain probed with CNTNAP2, CASK, and β-tubulin. CNTNAP2 and CASK, but not β-tubulin, are found in the washed membrane fraction (S5; red box). (j) Cropped western blot of time course and (k) quantification of CASK expression in cultured cortical neurons (n = 3 independent experiments). Values are means ± SEM. * P≤0.05, ** P≤0.01, *** P≤0.001; one-way ANOVA with Bonferroni’s correction (k, top graph; e). Student’s t-test (middle and bottom graphs; e).

    Article Snippet: Expression Time Course Cortices from CD1 WT mice aged P0, P14, P28, 4 months, and 6 months (sex not determined at P0, P14; males for P28, 4 months, 6 months) were homogenized in immunoprecipitation buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100 with Roche protease inhibitor cocktail) and solubilized for 1 hour at 4°C.

    Techniques: Expressing, Construct, Western Blot, Immunoprecipitation, Transfection, Proximity Ligation Assay, Staining, Cell Culture

    In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with anti-FLAG antibodies, resolved by SDS-PAGE, and detected by western blotting using the M2 anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.

    Journal: Journal of viral hepatitis

    Article Title: Sequences in the terminal protein and reverse transcriptase domains of the Hepatitis B Virus polymerase contribute to RNA binding and encapsidation

    doi: 10.1111/jvh.12225

    Figure Lengend Snippet: In vitro RNA binding activity of full-length HBV with mutant T3 and RT1 motifs (a) Accumulation of HBV P mutants. HBV P derivatives were expressed in transfected 293T cells, immunoprecipitated with anti-FLAG antibodies, resolved by SDS-PAGE, and detected by western blotting using the M2 anti-FLAG antibody; the exposure of the left gel was shorter to limit saturation of the more intense bands. The position of HBV P (P) and the antibody heavy chain (HC) are indicated. * denotes the position of an N-terminal fragment of the 3xFLAG-tagged wild-type P. (b) The immunoaffinity-purified HBV P derivatives were incubated with 32 P-labeled wild-type Hε or mutant Hε-dB RNA and co-precipitated products were resolved by SDS–PAGE. Input representing 0.5% of the indicated ε RNA added to each binding reaction mixture is in lanes 10, 11, 20, and 21. (c) Bound 32 P-labeled ε RNA signals were quantified via phosphorimaging and compared to the binding of wild-type P to Hε RNA. The data represent the mean ± one standard deviation from at least three independent experiments.

    Article Snippet: The FLAG lysis buffer was removed from aliquots of P-bound M2 beads, and then aliquots of beads were incubated with 0.5 μg 32 P-labeled ε RNAs in radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris (pH 7.0), 150 mM NaCl, 1 mM EDTA, 0.05% NP-40] with 1× complete protease inhibitor cocktail (Roche), 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 U/μl RNasin Plus RNase inhibitor (Promega) ( ).

    Techniques: In Vitro, RNA Binding Assay, Activity Assay, Mutagenesis, Transfection, Immunoprecipitation, SDS Page, Western Blot, Purification, Incubation, Labeling, Binding Assay, Standard Deviation

    Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery. A , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting ( IB ) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8.  Asterisk  denotes the heavy chain of the antibody used for IP.  Solid  and  open arrowheads  indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments.  B , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 ( WT ) or the K273A TRAIL-R1 mutant ( K / A ), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments.  C , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for  panel A. Asterisk  denotes the heavy chain of the antibody used for IP.  Solid  and  open arrowheads  indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments.  D , alignment of primary amino acid sequence of part of the transmembrane segment ( italic ) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in  E . Relevant potential ubiquitination sites are shown in  bold. E , MCF-7 Casp-3  cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in  D . TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments.  Asterisk  denotes the heavy chain of the antibody used for IP.

    Journal: The Journal of Biological Chemistry

    Article Title: Ubiquitination by the Membrane-associated RING-CH-8 (MARCH-8) Ligase Controls Steady-state Cell Surface Expression of Tumor Necrosis Factor-related Apoptosis Inducing Ligand (TRAIL) Receptor 1 *

    doi: 10.1074/jbc.M112.448209

    Figure Lengend Snippet: Steady-state ubiquitination of TRAIL-R1 on lysine residue 273 by an endogenous machinery. A , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). MARCH-8.HA cDNA (+) or an empty control vector (−) were additionally transfected as indicated. Cells were lysed in Nonidet P-40 buffer, TRAIL-R1 was isolated with anti(α)-mRFP antibody and immunoprecipitates ( IP ) were analyzed by immunoblotting ( IB ) with α-mRFP antibody to detect TRAIL-R1, α-FLAG antibody to detect ubiquitin and α-HA antibody to detect MARCH-8. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. Blot is representative of 4 independent experiments. B , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with either mRFP only (−), with mRFP chimeras of WT TRAIL-R1 ( WT ) or the K273A TRAIL-R1 mutant ( K / A ), or with a truncated TRAIL-R1 lacking the C-terminal 116 residues (ΔWT). TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and with α-FLAG antibody to detect ubiquitin. Data shown are representative of two independent experiments. C , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP only (−), or with mRFP-chimeras of WT TRAIL-R1 ( WT ) or its K273A lysine mutant ( K / A ). Cells were lysed by boiling in SDS, Nonidet P-40 buffer was added in excess and immunoprecipitation of TRAIL-R1 and analysis were performed as outlined for panel A. Asterisk denotes the heavy chain of the antibody used for IP. Solid and open arrowheads indicate, respectively, TRAIL-R1.mRFP and mRFP only. The blot is representative of 2 independent experiments. D , alignment of primary amino acid sequence of part of the transmembrane segment ( italic ) and the remaining 14 residues of the cytoplasmic tail of the truncated TRAIL-R1 mutants used in E . Relevant potential ubiquitination sites are shown in bold. E , MCF-7 Casp-3 cells were transfected to express FLAG-ubiquitin, together with mRFP-tagged TRAIL-R1 WT or mutants shown in D . TRAIL-R1 was isolated with α-mRFP antibody and immunoprecipitates were analyzed by immunoblotting with α-mRFP antibody to detect TRAIL-R1 and α-FLAG antibody to detect ubiquitin. The blot is representative of 2 independent experiments. Asterisk denotes the heavy chain of the antibody used for IP.

    Article Snippet: Western Blotting and Immunoprecipitation Cells were harvested and lysed in Nonidet P-40 buffer consisting of 50 mm Tris-HCl, pH 7.4, 1% Nonidet P-40, 150 mm NaCl, 1 mm PMSF and Complete Protease Inhibitors (Roche).

    Techniques: Transfection, Mutagenesis, Plasmid Preparation, Isolation, Immunoprecipitation, Sequencing

    Localization of TcdE in E. coli and C. difficile . A. Cytoplasmic and membrane proteins analysis of E. coli lysogens of λCmrΔ(SR) carrying pBR322 (control) or pCD463 (+ tcdE -6xHis). B. Cytoplasmic and membrane proteins analysis of C. difficile strain carrying either pMTL84151 (control) or pRG46 (+ tcdE -6xHis). (1) SDS-PAGE coomassie stained gel. Western blots probed with 6XHis Tag antibody (2), ATPase Beta subunit antibody (3), and Ribosomal subunits LI/L2 monoclonal antibody (4). C. Membrane protein samples from bacterial cells expressing TcdE-6His resuspended in denature or native sample buffers and analyzed by Western blot using His-Tag antibody. D. Membrane proteins of JIR8094, TcdE mutant and complemented TcdE mutant strains were harvested from bacterial cultures induced with 20 ng/ml of ATc for 2 hours, separated in 16% Tris-Glycine gel and transferred into PVDF membrane. Panels 1. Ponceau stained membrane; 2. Probed with TcdE antibody.

    Journal: PLoS Pathogens

    Article Title: Secretion of Clostridium difficile Toxins A and B Requires the Holin-like Protein TcdE

    doi: 10.1371/journal.ppat.1002727

    Figure Lengend Snippet: Localization of TcdE in E. coli and C. difficile . A. Cytoplasmic and membrane proteins analysis of E. coli lysogens of λCmrΔ(SR) carrying pBR322 (control) or pCD463 (+ tcdE -6xHis). B. Cytoplasmic and membrane proteins analysis of C. difficile strain carrying either pMTL84151 (control) or pRG46 (+ tcdE -6xHis). (1) SDS-PAGE coomassie stained gel. Western blots probed with 6XHis Tag antibody (2), ATPase Beta subunit antibody (3), and Ribosomal subunits LI/L2 monoclonal antibody (4). C. Membrane protein samples from bacterial cells expressing TcdE-6His resuspended in denature or native sample buffers and analyzed by Western blot using His-Tag antibody. D. Membrane proteins of JIR8094, TcdE mutant and complemented TcdE mutant strains were harvested from bacterial cultures induced with 20 ng/ml of ATc for 2 hours, separated in 16% Tris-Glycine gel and transferred into PVDF membrane. Panels 1. Ponceau stained membrane; 2. Probed with TcdE antibody.

    Article Snippet: C. difficile toxin and Lactate dehydrogenase (LDH) assays Culture supernatants were collected and filtered, and the cell pellets were resuspended in 10 mM Tris buffer, pH 8.0 containing a protease inhibitor cocktail (Roche, Mannheim, Germany).

    Techniques: SDS Page, Staining, Western Blot, Expressing, Mutagenesis