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    Structured Review

    New England Biolabs nebuffer 2
    Nebuffer 2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs haeiii
    The mutational effects in the laboratory drift correlate with sequence exchanges in <t>M.HaeIII</t> orthologs. a . The positional rates of evolution in M.HaeIII’s natural orthologs (‘Rate4site’, μ; red line) were plotted alongside the positional W rel values in M.HaeIII (blue line). The positional W rel values correspond to the average W rel ∑ i { W r e l i ⋅ log 2 [ 1 + 10 ⋅ f ( G 17 i ) ] } , Where i refers to all the possible single nucleotide mutations at a given residue position. Upper panel –positions 2 to 175; Lower panel –positions 176 to 330. Noted are M.HaeIII’s key functional residues, those of the cofactor binding site, the catalytic residues including the enzyme’s reaction center (Cys71, black arrow), and the target <t>DNA</t> binding residues. Also noted are positions of compensatory mutations that were enriched in the drift W rel > 1.1, listed in S2 Table ). b. M.HaeIII’s three-dimensional structure illustrated as a cartoon (PDB id 1dct). Residues are colored from blue to red according to their averaged W rel values (as in c ). The cofactor, SAM, is in yellow, and the enzyme’s catalytic residue (Cys71) is in green. c. The same as b for the positional diversity calculated by Rate4site (μ, as in c ) [ 65 ]. d. The distribution of PROVEAN scores for all possible single nucleotide missense mutations (n = 1,957). Shown are the distribution of mutations categorized as ‘deleterious’ ( W rel ≤0.6), and of mutations categorized as ‘nearly-neutral’, ‘neutral’ and ‘beneficial’ ( W rel > 0.6). e. The same distribution while excluding ‘nearly-neutral’ mutations.
    Haeiii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    New England Biolabs rna ligase buffer
    TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated <t>RNA</t> (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the <t>“+RppH”</t> pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule
    Rna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    New England Biolabs xho i restriction buffer
    Fig. 6. Xer recombination at psi and psiDC in the presence of XerC[De]. ( A ) Recombination of p- psi.psi and p- psiDC.psiDC in the presence of XerD and either XerC[De] or XerC. ( B ) Time course of recombination on p- psiDC.psiDC with XerC[De] and XerD. Reactions contained 40% glycerol and were cleaved with <t>Xho</t> I. Bands are indicated as follows: HJ, HJ intermediates; S, substrate fragments; P, product fragments.
    Xho I Restriction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs protein kinase ckii buffer
    Enhanced binding of βarr1 to phosphorylated Dvl. ( A ) HEK-293 cells were transfected with Myc-Dvl2 and FLAG-βarr1, and cell extracts were immunoprecipitated with anti-Myc antibody directed against Dvl2. Immunoprecipitated Dvl2 was phosphorylated in the presence of [γ- 32 P]ATP by endogenous Dvl2-associated kinase(s). Immunoprecipitated Dvl2 was processed for autoradiography ( Upper ) or incubated with His-βarr1 for > 3 h at 4°C. Washed Dvl2 immunoprecipitates were analyzed by immunoblot with anti-His antibody to detect associated βarr1 ( Lower ). ( B ) <t>MBP-Dvl1</t> was phosphorylated by protein kinase <t>CKII</t> in the presence of ATP. Phosphorylated and unphosphorylated control MBP-Dvl1 was incubated with His-βarr1 for > 3 h at 4°C, and associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1. ( C ) MBP-Dvl1 was phosphorylated to various stoichiometric levels by protein kinase CKII. Phosphorylated MBP-Dvl1 was then either eluted in sample buffer and processed for autoradiography ( Inset Upper ) or incubated with His-βarr1. MBP-Dvl1-associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1 ( Inset Lower ). The relative amounts of βarr1 bound to MBP-Dvl1 phosphorylated by protein kinase CKII were quantified as fold increases over that bound to unphosphorylated MBP-Dvl1. Results are the mean ± SEM of three independent experiments.
    Protein Kinase Ckii Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The mutational effects in the laboratory drift correlate with sequence exchanges in M.HaeIII orthologs. a . The positional rates of evolution in M.HaeIII’s natural orthologs (‘Rate4site’, μ; red line) were plotted alongside the positional W rel values in M.HaeIII (blue line). The positional W rel values correspond to the average W rel ∑ i { W r e l i ⋅ log 2 [ 1 + 10 ⋅ f ( G 17 i ) ] } , Where i refers to all the possible single nucleotide mutations at a given residue position. Upper panel –positions 2 to 175; Lower panel –positions 176 to 330. Noted are M.HaeIII’s key functional residues, those of the cofactor binding site, the catalytic residues including the enzyme’s reaction center (Cys71, black arrow), and the target DNA binding residues. Also noted are positions of compensatory mutations that were enriched in the drift W rel > 1.1, listed in S2 Table ). b. M.HaeIII’s three-dimensional structure illustrated as a cartoon (PDB id 1dct). Residues are colored from blue to red according to their averaged W rel values (as in c ). The cofactor, SAM, is in yellow, and the enzyme’s catalytic residue (Cys71) is in green. c. The same as b for the positional diversity calculated by Rate4site (μ, as in c ) [ 65 ]. d. The distribution of PROVEAN scores for all possible single nucleotide missense mutations (n = 1,957). Shown are the distribution of mutations categorized as ‘deleterious’ ( W rel ≤0.6), and of mutations categorized as ‘nearly-neutral’, ‘neutral’ and ‘beneficial’ ( W rel > 0.6). e. The same distribution while excluding ‘nearly-neutral’ mutations.

    Journal: PLoS Computational Biology

    Article Title: Systematic Mapping of Protein Mutational Space by Prolonged Drift Reveals the Deleterious Effects of Seemingly Neutral Mutations

    doi: 10.1371/journal.pcbi.1004421

    Figure Lengend Snippet: The mutational effects in the laboratory drift correlate with sequence exchanges in M.HaeIII orthologs. a . The positional rates of evolution in M.HaeIII’s natural orthologs (‘Rate4site’, μ; red line) were plotted alongside the positional W rel values in M.HaeIII (blue line). The positional W rel values correspond to the average W rel ∑ i { W r e l i ⋅ log 2 [ 1 + 10 ⋅ f ( G 17 i ) ] } , Where i refers to all the possible single nucleotide mutations at a given residue position. Upper panel –positions 2 to 175; Lower panel –positions 176 to 330. Noted are M.HaeIII’s key functional residues, those of the cofactor binding site, the catalytic residues including the enzyme’s reaction center (Cys71, black arrow), and the target DNA binding residues. Also noted are positions of compensatory mutations that were enriched in the drift W rel > 1.1, listed in S2 Table ). b. M.HaeIII’s three-dimensional structure illustrated as a cartoon (PDB id 1dct). Residues are colored from blue to red according to their averaged W rel values (as in c ). The cofactor, SAM, is in yellow, and the enzyme’s catalytic residue (Cys71) is in green. c. The same as b for the positional diversity calculated by Rate4site (μ, as in c ) [ 65 ]. d. The distribution of PROVEAN scores for all possible single nucleotide missense mutations (n = 1,957). Shown are the distribution of mutations categorized as ‘deleterious’ ( W rel ≤0.6), and of mutations categorized as ‘nearly-neutral’, ‘neutral’ and ‘beneficial’ ( W rel > 0.6). e. The same distribution while excluding ‘nearly-neutral’ mutations.

    Article Snippet: About 106 individual transformants were obtained in each round. (ii ) Colonies grown at 37°C overnight were combined, plasmid DNA was extracted and digested with HaeIII (10–20 units, in 50 μl of NEB buffer 2, for 2 hours at 37°C), and re-purified (PCR purification kit, QIAGEN). (iii ) The recovered plasmid DNA was re-transformed for another round of enrichment.

    Techniques: Sequencing, Functional Assay, Binding Assay

    TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated RNA (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the “+RppH” pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule

    Journal: BMC Genomics

    Article Title: TSS-EMOTE, a refined protocol for a more complete and less biased global mapping of transcription start sites in bacterial pathogens

    doi: 10.1186/s12864-016-3211-3

    Figure Lengend Snippet: TSS-EMOTE flowchart. The TSS-EMOTE assay consists of a wet-lab library preparation (panels a to g ) and in silico analyses (panel H to N). An asterisk continually marks the original 5’-base of tri-phosphorylated RNA (thin red line). a Total RNA is purified, and digested with XRN1 5’-exonuclease, which removes the vast majority of 5’ mono-phosphorylated RNA from the sample (including 16S and 23S rRNA). b and c The XRN1 treated RNA is mixed with large excess of a synthetic RNA oligo (Rp6, shown in blue), and split into two pools. Both pools receive T4 RNA ligase, but only the “+RppH” pool is co-treated with RppH, an enzyme that converts 5’ tri-phosphorylated ends to mono-phosphorylated ends, thus allowing the ligase to use them as substrates. d and e After the ligation reaction, a semi-random primer is used to reverse-transcribe the RNA and simultaneously add a 2.0 Illumina adapter (black “B”). This results in cDNA with a 2.0 Illumina adaptor (for reverse reads in paired-end sequencing) at the 5’-end and if the template RNA was ligated to an Rp6 oligo, then the cDNA will also have a complementary sequence to Rp6 at the 3’-end (cRp6). f PCR is used to specifically amplify cDNAs that carry the 2.0 Illumina adaptor and cRp6 sequences. This step moreover adds a 1.0 Illumina adaptor (for forward reads in paired-end sequencing) and a sample-specific 4-base EMOTE barcode (blue line and “XXX”, respectively) to index the molecules (different barcodes for the -RppH and + RppH pools). The barcode of the -RppH pool will designate molecules where the XRN1 treatments has been incomplete, and this information is incorporated into the in silico analysis (see below). g The barcoded DNA from various samples (and pools) can be mixed, and loaded directly into an Illumina HiSeq machine. Millions of 50 nt sequences are obtained, each of which will span the EMOTE barcode, both known and random sections of the Rp6 oligo (see Methods ), and it will reveal the first 20 nt of the native 5’-end of the ligated RNA molecule. These 20 nt are sufficient to map the vast majority of 5’-ends to a unique position on the small genomes of the bacteria in this study. However, longer Illumina reads (and thus longer mapping sequences) can be used if the TSSs are in repeated regions or if large-genome organisms, such as humans, are being examined. h The in silico pipeline input consists of stranded RNA-seq reads for one or multiple biological replicates in FASTQ format. Each replicate includes a FASTQ for the -RppH pool and another for the + RppH pool. i The FASTQ files go through EMOTE-conv software [ 51 ] that parses the reads, aligns them to the genome, and perform the quantification. Thus, for each genomic position we obtain the number of reads whose first nucleotide align at this genomic position, and on which strand it maps. The counts are further corrected for PCR biases by looking at the unique molecular identifiers (UMIs) sequences available in the unaligned part of the EMOTE read. j Quantification counts obtained for + RppH and -RppH pools are compared through a beta-binomial model that tests whether the identified 5’ ends in the + RppH pool is significantly enriched over the identified 5’ ends in the -RppH pool at a given position. The process results in a p-value that reflects our confidence in the genomic position to be enriched in the + RppH pool of the biological replicate. k The p-values of all the biological replicates are combined into a single p-value with Fisher’s method. l and m To correct the p-values for multiple testing across all genomic positions, the false discovery rate (FDR) is evaluated and only those with a FDR ≤ 0.01 are considered to be TSSs. Note also that for the FDR is only calculated for genomic positions with at least 5 detected ligation-events in at least one of the + RppH pools (UMI ≥ 5). n The TSSs then enter an annotation process that retrieve their surrounding sequence and downstream ORFs. TSSs separated by less than 5 bp are clustered together. Finally, to draw a global picture of operon structures, an independent detection of transcription terminators is operated with the software TransTermHP [ 39 ]. o Sequence of the RNA oligo Rp6 and a typical Illumina sequencing read from a TSS-EMOTE experiment. The Recognition Sequence serves as priming site for the PCR in panel F. UMI: The randomly incorporated nucleotides in the Rp6 oligo that serves to whether Illumina reads with identical Mapping Sequences originate from separate ligation events. CS: Control Sequence. EB: EMOTE barcode to index the Illumina reads. An asterisk indicates the 5’ nucleotide of the original RNA molecule

    Article Snippet: The “-RppH mix” consisted of 3.5 μl water, 2 μl RNA ligase buffer, 2 μl 10 mM ATP, 1 μl Murine RNase inhibitor (New England Biolabs), and 2 μl RNA ligase 1 (New England Biolabs).

    Techniques: In Silico, Purification, Ligation, Sequencing, Polymerase Chain Reaction, RNA Sequencing Assay, Software

    Fig. 6. Xer recombination at psi and psiDC in the presence of XerC[De]. ( A ) Recombination of p- psi.psi and p- psiDC.psiDC in the presence of XerD and either XerC[De] or XerC. ( B ) Time course of recombination on p- psiDC.psiDC with XerC[De] and XerD. Reactions contained 40% glycerol and were cleaved with Xho I. Bands are indicated as follows: HJ, HJ intermediates; S, substrate fragments; P, product fragments.

    Journal: The EMBO Journal

    Article Title: Accessory factors determine the order of strand exchange in Xer recombination at psi

    doi: 10.1093/emboj/cdf379

    Figure Lengend Snippet: Fig. 6. Xer recombination at psi and psiDC in the presence of XerC[De]. ( A ) Recombination of p- psi.psi and p- psiDC.psiDC in the presence of XerD and either XerC[De] or XerC. ( B ) Time course of recombination on p- psiDC.psiDC with XerC[De] and XerD. Reactions contained 40% glycerol and were cleaved with Xho I. Bands are indicated as follows: HJ, HJ intermediates; S, substrate fragments; P, product fragments.

    Article Snippet: The other lane was excised from the gel and equilibrated for 2 h with two changes in Xho I restriction buffer (New England Biolabs NEBuffer 2 + BSA).

    Techniques:

    Enhanced binding of βarr1 to phosphorylated Dvl. ( A ) HEK-293 cells were transfected with Myc-Dvl2 and FLAG-βarr1, and cell extracts were immunoprecipitated with anti-Myc antibody directed against Dvl2. Immunoprecipitated Dvl2 was phosphorylated in the presence of [γ- 32 P]ATP by endogenous Dvl2-associated kinase(s). Immunoprecipitated Dvl2 was processed for autoradiography ( Upper ) or incubated with His-βarr1 for > 3 h at 4°C. Washed Dvl2 immunoprecipitates were analyzed by immunoblot with anti-His antibody to detect associated βarr1 ( Lower ). ( B ) MBP-Dvl1 was phosphorylated by protein kinase CKII in the presence of ATP. Phosphorylated and unphosphorylated control MBP-Dvl1 was incubated with His-βarr1 for > 3 h at 4°C, and associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1. ( C ) MBP-Dvl1 was phosphorylated to various stoichiometric levels by protein kinase CKII. Phosphorylated MBP-Dvl1 was then either eluted in sample buffer and processed for autoradiography ( Inset Upper ) or incubated with His-βarr1. MBP-Dvl1-associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1 ( Inset Lower ). The relative amounts of βarr1 bound to MBP-Dvl1 phosphorylated by protein kinase CKII were quantified as fold increases over that bound to unphosphorylated MBP-Dvl1. Results are the mean ± SEM of three independent experiments.

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

    Article Title: ?-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated dishevelled proteins

    doi: 10.1073/pnas.211572798

    Figure Lengend Snippet: Enhanced binding of βarr1 to phosphorylated Dvl. ( A ) HEK-293 cells were transfected with Myc-Dvl2 and FLAG-βarr1, and cell extracts were immunoprecipitated with anti-Myc antibody directed against Dvl2. Immunoprecipitated Dvl2 was phosphorylated in the presence of [γ- 32 P]ATP by endogenous Dvl2-associated kinase(s). Immunoprecipitated Dvl2 was processed for autoradiography ( Upper ) or incubated with His-βarr1 for > 3 h at 4°C. Washed Dvl2 immunoprecipitates were analyzed by immunoblot with anti-His antibody to detect associated βarr1 ( Lower ). ( B ) MBP-Dvl1 was phosphorylated by protein kinase CKII in the presence of ATP. Phosphorylated and unphosphorylated control MBP-Dvl1 was incubated with His-βarr1 for > 3 h at 4°C, and associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1. ( C ) MBP-Dvl1 was phosphorylated to various stoichiometric levels by protein kinase CKII. Phosphorylated MBP-Dvl1 was then either eluted in sample buffer and processed for autoradiography ( Inset Upper ) or incubated with His-βarr1. MBP-Dvl1-associated βarr1 was detected by immunoblot with anti-His antibody directed against βarr1 ( Inset Lower ). The relative amounts of βarr1 bound to MBP-Dvl1 phosphorylated by protein kinase CKII were quantified as fold increases over that bound to unphosphorylated MBP-Dvl1. Results are the mean ± SEM of three independent experiments.

    Article Snippet: MBP-Dvl1 was washed with protein kinase CKII buffer (20 mM Tris, pH 7.5/50 mM KCl/2 mM MgCl2 ) and phosphorylated with protein kinase CKII (New England Biolabs) and [γ-32 P]ATP (60 μM; final concentration, 400 cpm/pmol) at 30°C for 30 min. Phosphorylated MBP-Dvl1 was visualized by exposure of dried gels to film.

    Techniques: Binding Assay, Transfection, Immunoprecipitation, Autoradiography, Incubation