Journal: BMC Genomics

Article Title: Improved transcription and translation with L-leucine stimulation of mTORC1 in Roberts syndrome

doi: 10.1186/s12864-015-2354-y

Figure Lengend Snippet: Ribosomal components and translation initiation complexes were present at low levels in RBS cells. a Western blotting showed that 40S small ribosome proteins RPS7 and RPS19, and 60S large ribosome proteins RPL5, RPL23, and RPL24 were decreased in ESCO2 mutant (M) compared to WT fibroblasts (WT) or corrected fibroblasts (C). b L-Leu supplement, but not D-Leu, partially rescued RPS7 and RPL24 protein levels, and reversed the elevation of eIF2α phosphorylation in RBS cells. α-Tubulin and eiF2α serve as loading controls. c m7-GTP coupled beads were used to pull down translation initiation complexes from whole cell lysates. 4EBP1 protein was strongly enriched in RBS cells, accompanied by less binding of eIF4G1, but this trend was partially reversed in RBS cells treated with L-Leu. eIF4E levels were not affected. d Antibodies to eIF4E were used to pull down translation initiation complexes. 4EBP1 was present at high levels in RBS cells, correlating with less eIF4G1 and the inhibition of translation initiation. L-Leu supplement promoted the assembly of the translation competent eIF4E complex. e Antibodies to eIF3B were used to pull down translation initiation complexes. eIF4E and eIF4G1 were present at lower levels in RBS cells, but this trend was partially reversed by L-Leu supplement

Article Snippet: Reagents were obtained from the following sources: antibodies to S6K1, 4EBP1, eIF4E, phospho-S51 eIF2α, eIF2α, eIF4G1 from Cell Signaling; antibodies to eIF3b (N20), α-tubulin, S6K, RPS7, RPS19, RPL5 and horseradish-peroxidase-labelled anti-mouse, anti-goat and anti-rabbit secondary antibodies from Santa Cruz Biotechnology; Anti-p70S6K1 (phospho T389) antibody from Abcam company; antibodies to RPL23 from Sigma company; antibodies to RPL24 from Genetex company.

Techniques: Western Blot, Mutagenesis, Phospho-proteomics, Binding Assay, Inhibition

Journal: The Journal of Clinical Investigation

Article Title: Dietary potassium stimulates Ppp1Ca-Ppp1r1a dephosphorylation of kidney NaCl cotransporter and reduces blood pressure

doi: 10.1172/JCI158498

Figure Lengend Snippet: ( A ) Immunoblot analysis of renal PP1, I1, and calcineurin in control mice (WT/WT SPAK), or mice with 1 (CA/–) or 2 (CA/CA) CA-SPAK alleles fed a control or high-potassium diet for 4 days. Each lane is from a separate mouse. ( B ) Quantitative summaries for A ; shown is the average protein abundance of PP1, I1, and calcineurin for mice of each genotype on the control diet (orange) or the high-potassium diet (red). Data are relative to control mice on the control diet. ( C ) PP1A localization in control mice harboring 2 WT SPAK alleles (WT/WT). Representative images show PP1A (red) in DCT1, identified by parvalbumin (PV) labeling (green), from WT mice randomized to the control or high-potassium diet for 4 days. Scale bars: 15 μm. Graph shows quantitative image analysis of total cellular PP1A intensity ( n = 4 mice/group, each data point represents the average intensity of >30 cells/mouse). * P < 0.05, by 2-tailed Student’s t test. ( D ) I1 localization in CA-SPAK mice harboring 2 CA-SPAK alleles (CA/CA). Representative images show I1 (red) in DCT1 from homozygous CA-SPAK (WT) mice randomized to the control or high-potassium diet for 4 days. Scale bars: 20 μm. Parvalbumin labeling (green) identified DCT1 (green arrowheads) from the cortical thick ascending limb (TAL), which also expressed I1. Graph shows quantitative image analysis of total cellular I1 intensity (4 mice/group, each data point represents the average intensity of >30 cells/mouse). Data are the mean ± SEM. * P < 0.05, by 2-tailed Student’s t test.

Article Snippet: Eluate was incubated with either (a) 10 units of Protein Phosphatase 1 Catalytic Subunit, α-isoform (MilliporeSigma, catalog P7937; 6123.08 units/mg) and 1 mM MnCl 2 in 1× PP1 reaction buffer (10 mM NaCl, 5 mM imidazole [pH 7.4], 0.2 mM DTT, 2.5‰Tween 20); (b) 25 ng PP2Aα (SignalChem, catalog P16-20BH) in 1× PP2 reaction buffer (25 mM HEPES [pH 7.2], 50 mM NaCl, 2.5 mM EDTA, 50 mM imidazole, 0.2% 2-mercaptolethanol, 65 ng/μL BSA); or (c) 10 units of PP3 (MilliporeSigma, catalog C1907), 1 mM MnCl 2 and 10 μg/mL calmodulin (MilliporeSigma, catalog P0270) in 1× PP3 reaction buffer (50 mM Tris-HCl [pH 7.0], 50 μM CaCl 2 , 50 μg/mL BSA) at 30°C for 30 minutes.

Techniques: Western Blot, Control, Labeling

Journal: The Journal of Clinical Investigation

Article Title: Dietary potassium stimulates Ppp1Ca-Ppp1r1a dephosphorylation of kidney NaCl cotransporter and reduces blood pressure

doi: 10.1172/JCI158498

Figure Lengend Snippet: ( A ) In vitro protein phosphatase (PP) assay. Representative immunoblots of p-NCC and t-NCC are shown for assays of NCC with PP1A (lanes 1 and 2), PP2A (lanes 5 and 6), and calcineurin A (CALNA) or vehicle (lanes 3, 7, and 11). For these studies, NCC was isolated by IP with an anti-NCC antibody (lanes 1, 2, 3, 5, 6, and 7) and compared with the negative control IgG (lanes 4, 8, and 12). ( B ) PP1A preferentially interacted with NCC in glutathione-agarose affinity chromatography assays with the GST fusion protein of the NCC terminus GST-NCC-(N), but not the negative control GST alone. Shown are representative immunoblot binding assays with recombinant PP1A, PP2A, and CALNA. Input protein phosphatase (lane 1) is shown relative to GST-bound (lane 2) or GST-NCC-(N) (lane 3). ( C ) A greater amount of PP1A co-immunoprecipitated with NCC when the extracellular K + concentration was high. Representative immunoblots show NCC, PP1A, and p-NCC (T58) in anti-FLAG immunoprecipitated samples relative to input from FLAG-tagged NCC-expressing MDCKI cells incubated with 3.5 mM (control), 0.5 mM (low), or 8 mM (high) K + buffers. Low-chloride buffer was used as a positive control to elevate p-NCC (T58) levels. Graphs show semiquantitative assessment of NCC, PP1, and p-NCC (T58) in NCC immunoprecipitated samples (bottom, right) relative to input. Data are from 3 individual experiments with 3 replicates for each condition ( n = 9). * P < 0.05, relative to the low-K + condition, by 1-way ANOVA followed by multiple-comparison test. Data are presented as the mean ± SEM. CALNA, calcineurin.

Article Snippet: Eluate was incubated with either (a) 10 units of Protein Phosphatase 1 Catalytic Subunit, α-isoform (MilliporeSigma, catalog P7937; 6123.08 units/mg) and 1 mM MnCl 2 in 1× PP1 reaction buffer (10 mM NaCl, 5 mM imidazole [pH 7.4], 0.2 mM DTT, 2.5‰Tween 20); (b) 25 ng PP2Aα (SignalChem, catalog P16-20BH) in 1× PP2 reaction buffer (25 mM HEPES [pH 7.2], 50 mM NaCl, 2.5 mM EDTA, 50 mM imidazole, 0.2% 2-mercaptolethanol, 65 ng/μL BSA); or (c) 10 units of PP3 (MilliporeSigma, catalog C1907), 1 mM MnCl 2 and 10 μg/mL calmodulin (MilliporeSigma, catalog P0270) in 1× PP3 reaction buffer (50 mM Tris-HCl [pH 7.0], 50 μM CaCl 2 , 50 μg/mL BSA) at 30°C for 30 minutes.

Techniques: In Vitro, Western Blot, Isolation, Negative Control, Affinity Chromatography, Binding Assay, Recombinant, Immunoprecipitation, Concentration Assay, Expressing, Incubation, Control, Positive Control, Comparison

Journal: Cell

Article Title: Transient protein-RNA interactions guide nascent ribosomal RNA folding

doi: 10.1016/j.cell.2019.10.035

Figure Lengend Snippet: (A) Temperature dependence of the long S7-bound lifetime phase shown for Δ270 truncation construct and the full 3’domain RNA in absence of any other r-proteins. The error bars represent the 95 % confidence intervals of the fit. (B) Nomura ribosome assembly map. (C) Experimental setup. (D) The probability of stable S7 incorporation increases with the presence of secondary binding r-proteins; temperature = 35 °C; [Cy5-S7] = 20 nM; [unlabeled r-proteins] = 400 nM, [S15] = 2 μM. (E and F) Simplified single-molecule traces for S7 binding at 35 °C in absence (E) or presence (F) of S9, S13 and S19. (G) Tertiary interactions between H41 and H42 are required for stable S7 binding. The area of the dots is proportional to the populations of the two lifetime phases. The error bars represent the 95 % confidence intervals of the fit. (H) Model on how RNA tertiary interactions and r-proteins progressively stabilize S7. Number of molecules analyzed in (D) (n) = 101, 120, 144, 115, 111, 141, 98, 149, 121, 57, 57. See also Figures S4, S5 and Data S2.

Article Snippet: Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains E. coli BL21-Gold (DE3) Agilent Cat#230132 E. coli Tuner ™ (DE3) Novagen Cat#70623-3 Chemicals, Peptides, and Recombinant Proteins Protocatechuic acid (PCA) Pacific Biosciences Cat#100-215-400 Protocatechuate-3,4-dioxygenase (PCD) Pacific Biosciences Cat#001-028-310 TSY Pacific Biosciences Cat#100-214-900 Cy5 Maleimide Mono-Reactive Dye 5-Pack GE Healthcare Cat#PA25031 Cy5.5 Sulfo-Cyanine5 maleimide Lumiprobe Cat#13380 Cy3 Maleimide Mono-Reactive Dye 5-Pack GE Healthcare Cat#PA23031 E. coli RNA polymerase holoenzyme NEB Cat#M0551S Biolipidure-203 NOF America Corporation Cat#Biolipidure-203 Biolipidure-206 NOF America Corporation Cat#Biolipidure-206 Purified ribosomal proteins of 16S rRNA 3’domain This paper N/A Oligonucleotides Primers, geneBlocks-based DNA transcription templates, labeled DNA oligonucleotides See Data S1 N/A ACU RNA trinucleotide Dharmacon N/A Recombinant DNA pRSF-1b Novagen Cat#71330-3 pRSF-1b-S2 This paper Addgene Plasmid #128590 pRSF-1b-S3 This paper Addgene Plasmid #128591 pRSF-1b-S7-S83C/delta157-178 This paper Addgene Plasmid #128592 pRSF-1b-S10 This paper Addgene Plasmid #128594 pRSF-1b-S13-C85S/P112C This paper Addgene Plasmid #128595 pRSF-1b-S14 This paper Addgene Plasmid #128596 pRSF-1b-S19-T48C This paper Addgene Plasmid #128597 pET-S9 This paper Addgene Plasmid #132948 pRSF-1b-S3-M129C This paper Addgene Plasmid #132949 pRSF-1b-S15-T79C This paper Addgene Plasmid #133048 Software and Algorithms MATLAB R2015a Matworks Inc http://matlab.com Open in a separate window KEY RESOURCES TABLE

Techniques: Construct, Binding Assay

Journal: Cell

Article Title: Transient protein-RNA interactions guide nascent ribosomal RNA folding

doi: 10.1016/j.cell.2019.10.035

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact. table ft1 table-wrap mode="anchored" t5 caption a7 REAGENT or RESOURCE SOURCE IDENTIFIER Bacterial and Virus Strains E. coli BL21-Gold (DE3) Agilent Cat#230132 E. coli Tuner ™ (DE3) Novagen Cat#70623-3 Chemicals, Peptides, and Recombinant Proteins Protocatechuic acid (PCA) Pacific Biosciences Cat#100-215-400 Protocatechuate-3,4-dioxygenase (PCD) Pacific Biosciences Cat#001-028-310 TSY Pacific Biosciences Cat#100-214-900 Cy5 Maleimide Mono-Reactive Dye 5-Pack GE Healthcare Cat#PA25031 Cy5.5 Sulfo-Cyanine5 maleimide Lumiprobe Cat#13380 Cy3 Maleimide Mono-Reactive Dye 5-Pack GE Healthcare Cat#PA23031 E. coli RNA polymerase holoenzyme NEB Cat#M0551S Biolipidure-203 NOF America Corporation Cat#Biolipidure-203 Biolipidure-206 NOF America Corporation Cat#Biolipidure-206 Purified ribosomal proteins of 16S rRNA 3’domain This paper N/A Oligonucleotides Primers, geneBlocks-based DNA transcription templates, labeled DNA oligonucleotides See Data S1 N/A ACU RNA trinucleotide Dharmacon N/A Recombinant DNA pRSF-1b Novagen Cat#71330-3 pRSF-1b-S2 This paper Addgene Plasmid #128590 pRSF-1b-S3 This paper Addgene Plasmid #128591 pRSF-1b-S7-S83C/delta157-178 This paper Addgene Plasmid #128592 pRSF-1b-S10 This paper Addgene Plasmid #128594 pRSF-1b-S13-C85S/P112C This paper Addgene Plasmid #128595 pRSF-1b-S14 This paper Addgene Plasmid #128596 pRSF-1b-S19-T48C This paper Addgene Plasmid #128597 pET-S9 This paper Addgene Plasmid #132948 pRSF-1b-S3-M129C This paper Addgene Plasmid #132949 pRSF-1b-S15-T79C This paper Addgene Plasmid #133048 Software and Algorithms MATLAB R2015a Matworks Inc http://matlab.com Open in a separate window KEY RESOURCES TABLE

Techniques: Recombinant, Purification, Labeling, Plasmid Preparation, Software