ybx1 recombinant protein  (Novus Biologicals)

Journal: JCI insight

Article Title: YBX1 promotes type H vessel-dependent bone formation in an m5C-dependent manner.

doi: 10.1172/jci.insight.172345

Figure Lengend Snippet: Figure 2. Endothelial Ybx1 deletion impairs CD31hiEMCNhi endothelium formation and bone formation. (A and B) RT-qPCR analysis of Ybx1 expression in CD31hiEMCNhi ECs (A) and BMSCs (B) from EC-specific Ybx1-knockout female mice (Ybx1iΔEC) and their littermate controls (Ybx1fl/fl). (C) FACS analysis dot plot of CD31hiEMCNhi ECs in each group. (D) Quantification of type H (left) and L (right) ECs from in each group. (E and F) Representative images (E) and quantitation (F) of CD31 (green) and EMCN (red) immunostained, 4-week-old Ybx1iΔEC and Ybx1fl/fl femora. (G) ELISA analysis of estradiol levels in each group. (H) Representative images (left) and quantitation (right) of VEGFA (red) immunostained in each group. (I and J) Representative μCT imag- es (I) and quantitative μCT analysis (J) of trabecular bone microarchitecture of 4-week-old Ybx1iΔEC and Ybx1fl/fl mice. (K) Representative images (left) and quantitation (right) of Osterix+ (green) immunostained in each group. (L) Representative images (left) and quantitation (right) of COL1 (green)

Article Snippet: For the RNA pulldown experiment, BMP4- and CD31-specific biotinylated probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and incubated with YBX1 recombinant protein (NBP2-30101, Novus) at 4°C for 3 hours.

Techniques: Quantitative RT-PCR, Expressing, Knock-Out, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: JCI insight

Article Title: YBX1 promotes type H vessel-dependent bone formation in an m5C-dependent manner.

doi: 10.1172/jci.insight.172345

Figure Lengend Snippet: Figure 4. YBX1 depletion leads to decreasing CD31 and EMCN stability in an m5C-dependent manner. (A) Genomic distribution of YBX1 CLIP-Seq peaks. (B) Venn diagram representing the overlap genes between YBX1 CLIP-Seq targets and YBX1-knockdown RNA-Seq targets. (C) Top 2 ranked sequence motifs enriched in YBX1 CLIP-Seq. (D–F) Genomic view of YBX1 binding to CD31, EMCN, and BMP4 loci. The frame area is 3′-UTR. (G) Semiquantitative PCR showed RBP immunoprecipitates using m5C RIP kit. (H and I) RNA pulldown analysis of binding between YBX1 protein and CD31 (or BMP4)–WT (or MUT)–probe. (J) Relative luciferase activity of HEK293T cells transfected with different pGL-4 vectors and pCMV-YBX1. (K–M) RT-qPCR analysis of the BMP4, CD31, and EMCN mRNA degradation rate of HUVECs treated with shYBX1 (blue lines) or shControl (red lines). (N) Western blotting analysis of the relative levels of CD31, BMP4, EMCN, and YBX1 protein expression. n = 3 independent experiments. Data are shown as the mean ± SEM. ***P < 0.001 by 1-way ANOVA. CDS, coding sequences.

Article Snippet: For the RNA pulldown experiment, BMP4- and CD31-specific biotinylated probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and incubated with YBX1 recombinant protein (NBP2-30101, Novus) at 4°C for 3 hours.

Techniques: Knockdown, RNA Sequencing, Sequencing, Binding Assay, Luciferase, Activity Assay, Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: JCI insight

Article Title: YBX1 promotes type H vessel-dependent bone formation in an m5C-dependent manner.

doi: 10.1172/jci.insight.172345

Figure Lengend Snippet: Figure 7. PEG-PLGA nanoparticles carrying sciadopitysin enhance angiogenesis-dependent bone formation in aged female mice. (A) Schematic diagram of treating aged female mice with sciadopitysin. (B and C) FACS analysis dot plot (B) and quantification (C) of CD31hiEMCNhi ECs from femora and tibia. (D) Western blotting analysis of YBX1 in CD31hiEMCNhi ECs (upper) and BMSCs (lower) from aged female mice injected with vehicle (PEG-PLGA nanoparticles) and PEG-PLGA nanoparticles carrying sciadopitysin (CD31 modified). (E and F) Representative μCT images (E) and quantitative μCT analysis (F) of trabecular bone microarchitecture of femora. (G and H) Representative images (G) and quantification (H) of calcein double labeling in femora. Scale bar 50 μm. (I–N) Representative images and quantification of H&E staining (I and J), Ocn staining (K and L), and TRAP staining (M and N) in trabecular bone surfaces. Scale bar 100 μm and 50 μm. (O and P) Representative images (O) and quantitation (P) of CD31 (green) and EMCN (red) immunostaining of tibia. (Q and R) Repre- sentative images (Q) and quantitation (R) of VEGFA-immunostained (green) tibia. (S and T) Representative images (S) and quantitation (T) of BMP4-immu- nostained (red) tibia. Femora and tibia were obtained from aged female mice injected with vehicle (PEG-PLGA nanoparticles) and PEG-PLGA nanoparticles carrying sciadopitysin (CD31 modified). Scale bar 100 μm. n = 7 mice in each group. n = 2 independent experiments. Data are shown as the mean ± SEM. **P < 0.01; ***P < 0.001 by Student’s t test.

Article Snippet: For the RNA pulldown experiment, BMP4- and CD31-specific biotinylated probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and incubated with YBX1 recombinant protein (NBP2-30101, Novus) at 4°C for 3 hours.

Techniques: Western Blot, Injection, Modification, Labeling, Staining, Quantitation Assay, Immunostaining

Journal: Cell metabolism

Article Title: Reducing Hypothalamic Stem Cell Senescence Protects against Aging-Associated Physiological Decline.

doi: 10.1016/j.cmet.2020.01.002

Figure Lengend Snippet: Figure 4. Hnscr Binds to YB-1 and Protects It from Degradation (A) Mass spectrometry identified YB-1 in proteins pulled-down by biotinylated Hnscr in htNSCs. (B) Biotinylated Hnscr retrieved YB-1 in htNSCs as detected by western blot.

Article Snippet: To screen naturally occurring small-molecule compounds that target and stabilize YB-1, we performed molecular docking and virtual screening between YB-1 and the natural small molecular compounds library of Target Molecule (Target Mol), which contains more than 6,000 compounds.

Techniques: Mass Spectrometry, Western Blot

Journal: Cell metabolism

Article Title: Reducing Hypothalamic Stem Cell Senescence Protects against Aging-Associated Physiological Decline.

doi: 10.1016/j.cmet.2020.01.002

Figure Lengend Snippet: Figure 5. Overexpression of YB-1 Reduces the Senescent Effect of Hnscr Loss (A) The mRNA levels of Hnscr and p16INK4A in htNSCs. The value of the expression in the WT mice was set at an arbitrary value = 1. (B) The mRNA levels of YB-1 and p16INK4A in htNSCs. The value of the expression in the scramble-treated htNSCs was set at an arbitrary value = 1. (C) The mRNA levels of p16INK4A and Yb-1 in htNSCs. The value of the expression in the WT mice without YB-1 overexpression was set at an arbitrary value = 1. (D) p16INK4A and YB-1 protein levels in Hnscr-deleted or WT htNSCs with or without YB-1 overexpressed. (E) The levels of Hnscr and Yb-1 in htNSCs of WT mice 1 month after viral injection. The value of the expression in the control vector injection mice was set at an arbitrary value = 1. (F–H) Representative images (n = 18 photographs from 3 of experiments) of neurospheres generated from mice 1 month after viral injection (F). Scale bar, 100 mm. Their quantitation is shown in (G); relative size in (H). (legend continued on next page)

Article Snippet: To screen naturally occurring small-molecule compounds that target and stabilize YB-1, we performed molecular docking and virtual screening between YB-1 and the natural small molecular compounds library of Target Molecule (Target Mol), which contains more than 6,000 compounds.

Techniques: Over Expression, Expressing, Injection, Control, Plasmid Preparation, Generated, Quantitation Assay

Journal: Cell metabolism

Article Title: Reducing Hypothalamic Stem Cell Senescence Protects against Aging-Associated Physiological Decline.

doi: 10.1016/j.cmet.2020.01.002

Figure Lengend Snippet: Figure 6. Theaflavin 3-gallate (TF2A) Acts as an Hnscr Mimic (A) The optimized homology modeling structure of mouse YB-1. The green ribbon indicated a main barrel-like structure that is composed of 6 reverse-laminated beta-sheets. The upper part of the barrel-like structure (presented as the wheat-colored thread) indicated a dynamic loop area. Between them was the prediction of small molecule binding pockets (presented as pink balls) that consisted of 12 hydrophobic amino acids. (B) The molecular structure of TF2A. (C) The optimized binding modes with lowest binding energy generated by AutoDock Vina and key residues for interaction between TF2A and mouse YB-1. (D) The expression of YB-1 in mouse htNSCs treated with TF2A in a series of doses, as indicated. (E) The protein levels of YB-1 in TF2A pre-treated mouse htNSCs treated with cycloheximide (CHX). (F) Western blot analysis of YB-1-associated ubiquitination in TF2A pre-treated mouse htNSCs treated with Mg132. (G) Top: hPSCs derived neurospheres immunostained with Nestin and SOX2. Scale bar, 100 mm (n = 15 photographs from 3 experiments). Bottom: hPSC-derived NSCs were induced to differentiate after 7 days of differentiation. The cells were immunostained with neuronal marker Tuj1, oligodendrocyte marker O4, and astrocyte marker GFAP. Scale bar, 100 mm (n = 15 photographs from 3 experiments). (H) The protein levels of YB-1 in hPSC-derived NSCs treated with TF2A in a series of dose as indicated. Top: representative images of western blot. Bottom: quantification analysis of YB-1 in relation to GAPDH. Data are shown as mean ± SEM. The cell experiments were repeated 3 times. *p < 0.05; **p < 0.01; ***p < 0.001 by two-tailed Student’s t test or one-way ANOVA.

Article Snippet: To screen naturally occurring small-molecule compounds that target and stabilize YB-1, we performed molecular docking and virtual screening between YB-1 and the natural small molecular compounds library of Target Molecule (Target Mol), which contains more than 6,000 compounds.

Techniques: Binding Assay, Generated, Expressing, Western Blot, Ubiquitin Proteomics, Derivative Assay, Marker, Two Tailed Test

Journal: Cell reports

Article Title: Single mRNP Analysis Reveals that Small Cytoplasmic mRNP Granules Represent mRNA Singletons.

doi: 10.1016/j.celrep.2019.09.018

Figure Lengend Snippet: Figure 2. Structure of IMP1 and YBX1 mRNP Granules (A) HeLa cells were stained with anti-IMP1 and anti-YBX1 antibodies followed by Alexa Fluor 488 (green) and Alexa Fluor 647 (red) secondary antibodies, respectively. (A1–A3) Overview of the cell. Scale bars, 5 mm. (A4–A6) Blow-up of IMP1 and YBX1 granules in the indicated area (white square) in (A3). Scale bar, 0.2 mm. (A7) P bodies depicted by DCP1a-EGFP in combination with IMP1 staining and A8, pHcRed-G3BP in stress granules in combination with IMP1 staining. (B) IMP1 (green, Alexa Fluor 488) and YBX1 (red, Alexa Fluor 647) immunostaining in combination with ACTB mRNA FISH (cyan) using 48 Quasar 570 dye-labeled oligonucleotides corresponding to the entire ACTB mRNA. (B1–B4) Overview of a HeLa cell. Scale bars, 5 mm. (B5–B9) blow up of ACTB mRNA and IMP1_YBX1 containing granules. Scale bars, 0.2 mm (B5) and 0.1 mm (B6–B9). (B10–B19) IMP1 (green, Alexa Fluor 488) and YBX1 (red, Alexa Fluor 647) immunostaining in combination with GAPDH mRNA FISH (cyan) using 48 Quasar 570 dye-labeled (cyan) oligonucleotides corresponding to the entire GAPDH mRNA. (B10–B14) Overview of a HeLa cell. Scale bars, 5 mm. (B15–B19) Blow-up of GAPDH mRNA and IMP1_YBX1 containing granules. Scale bars, 0.2 mm (B15) and 0.1 mm (B16–B19). (B20 and B21) Double FISH with ACTB mRNA (red, Quasar 670-conjugated probes) and GAPDH mRNA (green, Quasar 570-conjugated probes) in combination with YBX1 immunostaining (gray, Alexa Fluor 488). (C) EGFP immunoprecipitation of transiently transfected HeLa cells with pEGFP-C1 (control) and pEGFP-IMP1. Immunodetection of GFP and GFP-IMP1, endogenous IMP1, YBX1, and GADPH, respectively, was performed in total lysate and immunoprecipitated (IP) fractions without () or with (+) RNase A treatment.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-IMP1 (raised against C-terminal peptide) Nielsen et al., 1999 N/A Goat polyclonal anti-IMP1 Santa Cruz Biotechnology Cat#E-20; RRID: AB_649425 Rabbit polyclonal anti-YBX1 Abcam Cat#ab12148; RRID: AB_2219278 Rabbit polyclonal anti-GAPDH Santa Cruz Biotechnology Cat#FL-335; RRID: AB_10167668 Mouse monoclonal anti-GFP Abcam Cat#ab1218; RRID: AB_298911 Mouse monoclonal anti-Nup153 Abcam Cat#ab24700; RRID: AB_2154467 Mouse monoclonal anti-PABPC1 Abcam Cat#ab6125; RRID: AB_2156878 Chemicals, Peptides, and Recombinant Proteins Recombinant human IMP1 Nielsen et al., 1999 N/A Recombinant human YBX1 Origene Cat#TP309835 Critical Commercial Assays Click-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay Kit Invitrogen Cat#C10456 Experimental Models: Cell Lines HeLa cell line ATCC CCL-2 Oligonucleotides siRNA against YBX1 30UTR 50 - GAACAGGCCUGGUGGGAAAUU - 30 (sense) 50 - UUUCCCACCAGGCCUGUUCUU 30 (antisense) Eurofins Genomics N/A Stellaris FISH Probes, Human ACTB with Quasar 570 Dye LGC Biosearch Cat#VSMF-2002-5 Stellaris FISH Probes, Human ACTB with Quasar 670 Dye LGC Biosearch Cat#VSMF-2003-5 Stellaris FISH Probes, Human GAPDH with Quasar 570 Dye LGC Biosearch Cat#SMF-2026-1 Recombinant DNA pEGFP-C1 Clontech N/A pEGFP-IMP1 This manuscript N/A pEGFP-IMP1_KH1-4mut This manuscript N/A pcDNA3.1+N-EGFP-YBX1 Genscript N/A pmCherry-YBX1 This manuscript N/A pmEos3.2-IMP1 This manuscript N/A PABPC1-GFP This manuscript N/A DCP1a-GFP This manuscript N/A pHcRed1-G3BP This manuscript N/A Software and Algorithms ZEN 2012 Carl Zeiss Microscopy N/A RStudio R Foundation N/A

Techniques: Staining, Immunostaining, Labeling, Immunoprecipitation, Transfection, Control, Immunodetection

Journal: Cell reports

Article Title: Single mRNP Analysis Reveals that Small Cytoplasmic mRNP Granules Represent mRNA Singletons.

doi: 10.1016/j.celrep.2019.09.018

Figure Lengend Snippet: Figure 3. IMP1 and YBX1 mRNP Formation at the Nuclear Pore HeLa cells were stained with anti-IMP1, anti-YBX1, and anti-NUP153 primary antibodies followed by Alexa Fluor 488 (green), Alexa 555 (cyan), and Alexa Fluor 647 (red) secondary antibodies, respectively. Moreover, the nucleus was stained with DAPI (deep blue). (A) Overview of a triple-stained cell and the area that is shown in the blow-up below. Scale bar, 2 mm. (B–E) Individual IMP1 (B), YBX1 (C), NUP153 (D), and DAPI (E) stainings. (F) Composite picture demonstrating the colocalization of NUP153 and the IMP1_YBX1 mRNP. Scale bar, 200 nm. Arrows indicate nuclear pores.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-IMP1 (raised against C-terminal peptide) Nielsen et al., 1999 N/A Goat polyclonal anti-IMP1 Santa Cruz Biotechnology Cat#E-20; RRID: AB_649425 Rabbit polyclonal anti-YBX1 Abcam Cat#ab12148; RRID: AB_2219278 Rabbit polyclonal anti-GAPDH Santa Cruz Biotechnology Cat#FL-335; RRID: AB_10167668 Mouse monoclonal anti-GFP Abcam Cat#ab1218; RRID: AB_298911 Mouse monoclonal anti-Nup153 Abcam Cat#ab24700; RRID: AB_2154467 Mouse monoclonal anti-PABPC1 Abcam Cat#ab6125; RRID: AB_2156878 Chemicals, Peptides, and Recombinant Proteins Recombinant human IMP1 Nielsen et al., 1999 N/A Recombinant human YBX1 Origene Cat#TP309835 Critical Commercial Assays Click-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay Kit Invitrogen Cat#C10456 Experimental Models: Cell Lines HeLa cell line ATCC CCL-2 Oligonucleotides siRNA against YBX1 30UTR 50 - GAACAGGCCUGGUGGGAAAUU - 30 (sense) 50 - UUUCCCACCAGGCCUGUUCUU 30 (antisense) Eurofins Genomics N/A Stellaris FISH Probes, Human ACTB with Quasar 570 Dye LGC Biosearch Cat#VSMF-2002-5 Stellaris FISH Probes, Human ACTB with Quasar 670 Dye LGC Biosearch Cat#VSMF-2003-5 Stellaris FISH Probes, Human GAPDH with Quasar 570 Dye LGC Biosearch Cat#SMF-2026-1 Recombinant DNA pEGFP-C1 Clontech N/A pEGFP-IMP1 This manuscript N/A pEGFP-IMP1_KH1-4mut This manuscript N/A pcDNA3.1+N-EGFP-YBX1 Genscript N/A pmCherry-YBX1 This manuscript N/A pmEos3.2-IMP1 This manuscript N/A PABPC1-GFP This manuscript N/A DCP1a-GFP This manuscript N/A pHcRed1-G3BP This manuscript N/A Software and Algorithms ZEN 2012 Carl Zeiss Microscopy N/A RStudio R Foundation N/A

Techniques: Staining

Journal: Cell reports

Article Title: Single mRNP Analysis Reveals that Small Cytoplasmic mRNP Granules Represent mRNA Singletons.

doi: 10.1016/j.celrep.2019.09.018

Figure Lengend Snippet: Figure 7. Molecular Composition of IMP1_YBX1 mRNP The number of IMP1, YBX1, and ACTB mRNA molecules in the mRNP were derived from localization microscopy (LM) or from FCS and compared with the average number of binding sites in the transcriptome from eCLIP and RNA immunoprecipitation sequencing analysis. For localization microscopy, cells were stained for IMP1, YBX1, and ACTB mRNA, and following bleaching, mRNP emitted photons were counted as described. (A) Examples of the localization microscopy images of YBX1 (red), IMP1 (green), and ACTB mRNA (cyan), respectively. Scale bar, 100 nm. (B) Counts per particle derived from FCS of cells transfected with GFP, GFP-IMP1_KH1-4mut, GFP-IMP1, GFP-YBX1, and with co-transfection of YBX1 30 UTR- directed siRNA and GFP-YBX1. Laser power was 0.02% in all measurements. (C) Summary of the data from localization microscopy, FCS, and binding sites predicted from either eCLIP (IMP1) or RNA immunoprecipitation sequencing (YBX1) experiments. (D) Immunofluorescence staining of PABPC1 (cyan, Alexa Fluor 488), IMP1 (green, Alexa Fluor 568), and YBX1 (red, Alexa Fluor 647). An overview of a whole HeLa cell is shown in the panel above (scale bar, 5 mm), and a blow-up image of the triple PABPC1, IMP1, and YBX1 staining of the area squared in the panel above is shown below (scale bar, 0.2 mm). Positioning of PABPC1 (cyan) in individual mRNPs depicted by YBX1 (red) staining is shown in the panel below (scale bar, 0.1 mm).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-IMP1 (raised against C-terminal peptide) Nielsen et al., 1999 N/A Goat polyclonal anti-IMP1 Santa Cruz Biotechnology Cat#E-20; RRID: AB_649425 Rabbit polyclonal anti-YBX1 Abcam Cat#ab12148; RRID: AB_2219278 Rabbit polyclonal anti-GAPDH Santa Cruz Biotechnology Cat#FL-335; RRID: AB_10167668 Mouse monoclonal anti-GFP Abcam Cat#ab1218; RRID: AB_298911 Mouse monoclonal anti-Nup153 Abcam Cat#ab24700; RRID: AB_2154467 Mouse monoclonal anti-PABPC1 Abcam Cat#ab6125; RRID: AB_2156878 Chemicals, Peptides, and Recombinant Proteins Recombinant human IMP1 Nielsen et al., 1999 N/A Recombinant human YBX1 Origene Cat#TP309835 Critical Commercial Assays Click-iT Plus OPP Alexa Fluor 488 Protein Synthesis Assay Kit Invitrogen Cat#C10456 Experimental Models: Cell Lines HeLa cell line ATCC CCL-2 Oligonucleotides siRNA against YBX1 30UTR 50 - GAACAGGCCUGGUGGGAAAUU - 30 (sense) 50 - UUUCCCACCAGGCCUGUUCUU 30 (antisense) Eurofins Genomics N/A Stellaris FISH Probes, Human ACTB with Quasar 570 Dye LGC Biosearch Cat#VSMF-2002-5 Stellaris FISH Probes, Human ACTB with Quasar 670 Dye LGC Biosearch Cat#VSMF-2003-5 Stellaris FISH Probes, Human GAPDH with Quasar 570 Dye LGC Biosearch Cat#SMF-2026-1 Recombinant DNA pEGFP-C1 Clontech N/A pEGFP-IMP1 This manuscript N/A pEGFP-IMP1_KH1-4mut This manuscript N/A pcDNA3.1+N-EGFP-YBX1 Genscript N/A pmCherry-YBX1 This manuscript N/A pmEos3.2-IMP1 This manuscript N/A PABPC1-GFP This manuscript N/A DCP1a-GFP This manuscript N/A pHcRed1-G3BP This manuscript N/A Software and Algorithms ZEN 2012 Carl Zeiss Microscopy N/A RStudio R Foundation N/A

Techniques: Derivative Assay, Microscopy, Binding Assay, RNA Immunoprecipitation, Sequencing, Staining, Transfection, Cotransfection

Journal: Journal of proteome research

Article Title: Discovery of Tamoxifen and N-Desmethyl Tamoxifen Protein Targets in MCF-7 Cells Using Large-Scale Protein Folding and Stability Measurements

doi: 10.1021/acs.jproteome.7b00442

Figure Lengend Snippet: High confidence TAM-induced protein stability hits.

Article Snippet: SPROX Protocol with Phenacyl Bromide Labelling (PAB-SPROX) A PAB-SPROX experiment was performed on a purified recombinant human YBX1 construct (Novus Biologicals, #NBP2-30101) both in the presence and absence of TAM according to a previously established protocol 23 .

Techniques: Multiplex sample analysis

Journal: Journal of proteome research

Article Title: Discovery of Tamoxifen and N-Desmethyl Tamoxifen Protein Targets in MCF-7 Cells Using Large-Scale Protein Folding and Stability Measurements

doi: 10.1021/acs.jproteome.7b00442

Figure Lengend Snippet: Pulse proteolysis with purified recombinant YBX1 +/− TAM. A) Pulse proteolysis of YBX1 +/− TAM in increasing concentration of urea. Red box depicting TAM-induced difference at 0.75 M Urea. B) Intact YBX1 and pulse proteolysis of YBX1 in the absence of TAM at low and high denaturant.

Article Snippet: SPROX Protocol with Phenacyl Bromide Labelling (PAB-SPROX) A PAB-SPROX experiment was performed on a purified recombinant human YBX1 construct (Novus Biologicals, #NBP2-30101) both in the presence and absence of TAM according to a previously established protocol 23 .

Techniques: Purification, Recombinant, Concentration Assay

Journal: Journal of proteome research

Article Title: Discovery of Tamoxifen and N-Desmethyl Tamoxifen Protein Targets in MCF-7 Cells Using Large-Scale Protein Folding and Stability Measurements

doi: 10.1021/acs.jproteome.7b00442

Figure Lengend Snippet: SPROX data generated on YBX1 +/− TAM. The filled circles represent data generated on the YBX1 peptide RPQYSNPPVQGEVMEGADNQGAGEQGRPVR in the PAB-SPROX analysis of the purified recombinant YBX1 construct. The open circles represent data on the oxidized version of the same YBX1 peptide in the SILAC-SPROX analysis of the unpurified protein in the MCF-7 cell lysate. Both data sets are consistent with a TAM-induced stabilization of YBX1.

Article Snippet: SPROX Protocol with Phenacyl Bromide Labelling (PAB-SPROX) A PAB-SPROX experiment was performed on a purified recombinant human YBX1 construct (Novus Biologicals, #NBP2-30101) both in the presence and absence of TAM according to a previously established protocol 23 .

Techniques: Generated, Purification, Recombinant, Construct, Multiplex sample analysis

Journal: Journal of proteome research

Article Title: Discovery of Tamoxifen and N-Desmethyl Tamoxifen Protein Targets in MCF-7 Cells Using Large-Scale Protein Folding and Stability Measurements

doi: 10.1021/acs.jproteome.7b00442

Figure Lengend Snippet: STRING analysis of the 27 high-confidence TAM and NDT hits. The STRING analysis was conducted using medium confidence data from experiments. Also included in the STRING analysis were the MAPK1, AKT1 and ESR1 (see text). The red dashed line represents an experimentally observed direct interaction between ER and YBX1 that was recently reported in reference.34

Article Snippet: SPROX Protocol with Phenacyl Bromide Labelling (PAB-SPROX) A PAB-SPROX experiment was performed on a purified recombinant human YBX1 construct (Novus Biologicals, #NBP2-30101) both in the presence and absence of TAM according to a previously established protocol 23 .

Techniques: