Journal: BBA Clinical

Article Title: Mutation of EMG1 causing Bowen–Conradi syndrome results in reduced cell proliferation rates concomitant with G2/M arrest and 18S rRNA processing delay

doi: 10.1016/j.bbacli.2014.05.002

Figure Lengend Snippet: Ribosomal subunit levels at steady-state are normal in BCS patient cells. (A, B) Sucrose gradient separation of 40S and 60S ribosomal subunits in lymphoblasts (A) and fibroblasts (B). Ribosomal subunits were detected following sucrose gradient separation by absorbance at 254 nm, and examples of the resultant tracings are shown. (C, D) Fractions were collected at regular intervals and probed by immunoblot analysis for ribosomal proteins using anti-RPS19 and anti-RPL7 antibodies to verify the separation of the small (RPS) and large (RPL) subunits. (E, F) The area under the curve was determined for individual peaks, and the ratio of large to small subunit was calculated. No significant difference between unaffected control and BCS-affected cells was found in lymphoblasts (E) and fibroblasts (F). The mean of six individual experiments and SEM are shown.

Article Snippet: Primary antibody dilutions were used as follows: RPL7 rabbit polyclonal at 1:5000 (Bethyl laboratories), RPS19 rabbit polyclonal at 1:3000 (Proteintech Group), TP53 2B2.68 mouse monoclonal at 1:2000 (Santa Cruz), p21/WAF1/Cip1 mouse monoclonal at 1:250 (Millipore), and β-actin mouse monoclonal at 1:5000 (Sigma).

Techniques: Western Blot, Control

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

Journal: Clinical and Translational Medicine

Article Title: MUC1 drives ferroptosis resistance in ICC via Src‐mediated FSP1 deubiquitination and myristoylation

doi: 10.1002/ctm2.70495

Figure Lengend Snippet: MUC1 interacts with and activates Src kinase to regulate FSP1 expression. (A) MUC1 interactome identification in HuCCT1 cells: left panel shows Coomassie blue staining of immunoprecipitated proteins; right panel displays tabulated IP–MS results of MUC1‐associated proteins. (B) Co‐immunoprecipitation assays demonstrating interaction between MUC1 and Src in HuCCT1 cells. (C) In vitro GST pull‐down assay confirming direct binding between recombinant MUC1 and Src proteins. (D) Immunofluorescence co‐localisation analysis of MUC1 (green) and Src (red) in HuCCT1 cells. Representative images shown at 600× magnification; scale bar = 10 µm. (E) Western blot analysis of Src phosphorylation at Tyr419 in HuCCT1 cells with MUC1 manipulation. (F) Western blot analysis of FSP1 protein levels in HuCCT1 cells with MUC1 overexpression treated with or without Src inhibitors. (G) Ferrorrange staining for ferrous ion detection in HuCCT1 cells under indicated treatments. Representative images shown at 200× magnification; scale bar = 25 µm. (H) Lipid peroxidation levels measured by MDA assay. (I) Flow cytometric analysis of intracellular ROS levels using DCFH‐DA staining. (J) Representative images of subcutaneous tumours from different treatment groups: control, RSL3 treatment and RSL3 + dasatinib combination therapy. (K) Quantitative analysis of tumour weights across treatment groups. Data are shown as the means ± SD, the significant level was identified by * p < .05; ** p < .01; *** p < .001.

Article Snippet: Recombinant active Src kinase (Sino Biological, Beijing, China) was incubated with purified USP10 or NMT1 proteins in kinase buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 2 mM DTT, 1 mM Na 3 VO 4 , 5 mM β‐glycerophosphate and 100 μM ATP) at 30°C for 30 min.

Techniques: Expressing, Staining, Immunoprecipitation, Protein-Protein interactions, In Vitro, Pull Down Assay, Binding Assay, Recombinant, Immunofluorescence, Western Blot, Phospho-proteomics, Over Expression, Multiple Displacement Amplification, Control

Journal: Clinical and Translational Medicine

Article Title: MUC1 drives ferroptosis resistance in ICC via Src‐mediated FSP1 deubiquitination and myristoylation

doi: 10.1002/ctm2.70495

Figure Lengend Snippet: Src phosphorylates USP10 to regulate FSP1 stability. (A) Co‐immunoprecipitation assays demonstrating interaction between Src and USP10 in HuCCT1 cells. (B) In vitro GST pull‐down assay confirming direct binding between recombinant Src and USP10 proteins. (C–F) Domain mapping experiments identifying critical interaction regions: (C) schematic of Src domain mutants, (D) GST pull‐down assay with Src mutants, (E) schematic of USP10 domain mutants, (F) GST pull‐down assay with USP10 mutants. (G) Phos‐tag SDS‐PAGE analysis of USP10 phosphorylation status following in vitro kinase reactions with purified Src, MUC1 and Src inhibitor. (H) Co‐immunoprecipitation assays examining Src–USP10 interaction in HuCCT1 cells with MUC1 knockdown.

Article Snippet: Recombinant active Src kinase (Sino Biological, Beijing, China) was incubated with purified USP10 or NMT1 proteins in kinase buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl2, 2 mM DTT, 1 mM Na 3 VO 4 , 5 mM β‐glycerophosphate and 100 μM ATP) at 30°C for 30 min.

Techniques: Immunoprecipitation, In Vitro, Pull Down Assay, Binding Assay, Recombinant, SDS Page, Phospho-proteomics, Purification, Knockdown