Why are proteins basophilic

We further propose that FILIP1-mediated homeostasis of filamins is required for the proper development of skeletal and cardiac muscle cells and likely also their maintenance under conditions of sustained mechanical stress.

Experiments were performed in three independent biological replicates with a label switch. Experiments were performed in six independent biological replicates and two technical replicates with label switches. Cells were regularly tested for mycoplasma contamination and found to be mycoplasma-negative.

In brief, cells were placed on ice and washed twice with ice-cold phosphate-buffered saline PBS , supplemented with 0. Reduction and alkylation of proteins was performed as described 9 with slight modifications. Three-minutes fractions 30 fractions per run were collected and each fraction was used for titanium dioxide TiO 2 -based enrichment of phosphopeptides.

Samples from three independent biological experiments were further processed for phosphopeptide enrichment in a randomized procedure. Phosphopeptides were also enriched from each of the supernatants using sequential metal oxide affinity chromatography SMOAC In brief, the supernatant and the first washing step from the TiO 2 enrichment procedure were combined and lyophilized overnight. Dried peptides were resolved in 0. The MS instruments were externally calibrated using standard compounds and equipped with a nanoelectrospray ion source and a stainless steel emitter Thermo Fischer Scientific.

Phosphopeptides were enriched as described above and mixed with For parallel reaction monitoring PRM , an inclusion list comprising 16 precursors with 96 transitions was generated with Skyline 2. One scan cycle consisted of a full MS1 scan at a resolution of 70,, an automatic gain control AGC target of 3e6 ions, a max.

Each PRM scan targeted one precursor of the inclusion list at a resolving power of 17,, an AGC target of 2e5, a max.

Cell lysis was performed as described Coupling of biotinylated proteins to streptavidin Dynabeads Invitrogen, Karlsruhe, Germany and subsequent washing steps were performed as described The precursor mass tolerance was set to 20 ppm for the first search and to 4.

Oxidation of methionine and phosphorylation of serine S , threonine T and tyrosine Y were set as variable modifications and cysteine carbamidomethylation as fixed modification. Subsequently, the intensity ratios of all localized sites were analyzed statistically at the log 2 -scale.

A linear model was applied to jointly estimate IGF-1 and LY treatment effects and for assessing their significance. In this analysis, two labeling effects were included into the linear model to adjust for a labeling bias.

The estimated residual variances of the individual proteins were regularized by averaging with the median variance of all proteins. False-discovery rates were calculated by the linear step-up procedure introduced by Benjamini and Hochberg. These peptides were further analyzed with the R motif-X package 53 to identify enriched motifs.

The default motif-X parameter, minimum motif occurrence of 10 and a p value cutoff of 1e-6 were used. For the visualization of the sequence logos, the Python package Logomaker 54 was used. For the kinase-substrate enrichment analysis KSEA 27 quantified phosphopeptides were annotated with their respective kinases according to the PhosphositePlus 55 kinase-substrate dataset downloaded July , filtered for kinase-substrate interactions reported in vivo for mice.

Kinase isoforms were grouped prior to calculating the kinase score and only kinases with at least 5 mapped substrates were regarded in the further analysis. For heatmap visualization row-wise z-score transformation was performed.

For analysis of MS data from in vitro kinase assays, raw files were processed using Andromeda embedded in MaxQuant 1. Precursor and fragment mass tolerances were set to 10 ppm and 0. MaxQuant msms. MS1 intensities were calculated using the MS1 filtering tutorial provided by the software developers. Orbitrap default parameters were used for transition settings.

Extracted ion chromatograms of imported peptides were manually inspected for correct peak picking and peak integration was adjusted, if necessary. Mean and standard error of the mean SEM were calculated for technical replicates and then for biological replicates.

Intensities of all phosphopeptides were summed and normalized by the respective summed intensity. Precursor and fragment mass tolerances were set to 5 ppm and 0. The precursor mass tolerance the first search was set to 20 ppm and to 4. The mass tolerance of fragment ions was set to 0. The protein groups file was processed using Perseus 1. Mean log 10 ratios were subjected to hierarchical clustering by Euclidean average k -means cluster analysis.

Gene Ontology GO analysis for cellular component release 30 Apr was performed using the Cytoscape 3. Precursor and fragment mass tolerances were set to 4.

Text mining for the identification of kinases potentially phosphorylating significantly regulated peptides was performed on publicly available biomedical literature sources, i. As of February , Medline contained over 30 million abstracts and the open access content of PubMed Central comprised over 2. Tools from the JCoRe repository were used for linguistic processing, performing tasks such as segmentation of each text into basic linguistic units sentences and words , acronym recognition to determine the long forms of abbreviation terms , and grammatical analysis For subsequent semantic analysis, GeNo tagger was used to identify gene and protein occurrences in text 61 followed by running the BioSem tool to identify molecular events between previously recognized gene and protein mentions Event items referencing members of significantly regulated phosphopeptides were extracted and further post-processed to obtain the final result list.

These identifiers were ultimately mapped to all associated EntrezGene identifiers resulting in an extended list of Entrez IDs, which, in turn, were used for querying event selection. These identifiers were ultimately mapped to all associated EntrezGene identifiers resulting in an extended list of Entrez IDs which, in turn, were used for event selection.

Expression and purification of recombinant His 6 - or GST-fusion proteins were performed according to standard protocols described before with minor modifications In brief, E.

Beads were transferred to a column and washed five times with 4. Subsequently, samples were analyzed by SDS-PAGE followed by quantitative Western blot analysis using antibodies directed against the respective tag of the protein. Peroxidase-conjugated goat anti-rat secondary antibodies Dianova were used to visualize signals by enhanced chemiluminesence.

The supernatant was used immediately for binding studies. FCS was performed with an instrumental setup described previously Fluorescence recovery after photobleaching FRAP experiments were performed and analyzed essentially as described previously 9 , In each myotube, 1—3 regions of interest ROIs, single Z-discs were bleached.

Mean halftimes were calculated on the basis of exchange process of bound protein with the soluble fraction in the slower phase of the biphasic recovery profile. Mobile fractions are calculated based on the recovery of the fluorescence in comparison to the initial starting intensity in the ROIs.

At specific time points after transfection, cells were lysed for Western Blot analysis or analyzed by fluorescence microscopy. For western blot analysis, cells were lysed using RIPA buffer and protein concentrations were equalized using the Bradford assay.

Fixed cells were incubated with T12 anti-titin antibody and Alexaconjugated goat-anti-mouse IgG1 secondary antibodies. Cell nuclei were stained by adding DAPI to the secondary antibodies. All other constructs used in this work were obtained by cloning PCR products amplified with primers including restriction sites. Amplicons were cloned in the appropriate vectors and used for expression in immortalized mouse myoblasts, HEK or L40 yeast cells, or in E. The serum was affinity-purified against the antigen and preabsorbed against the carboxy-terminus of the highly homologous FILIP1L to avoid cross-reactivity and used in dilution.

Horseradish peroxidase HRP -conjugated anti-rabbit, anti-mouse, anti-rat and anti-sheep immunoglobulins were used in , dilution if not otherwise stated and purchased from Sigma-Aldrich, Thermo and Dianova Hamburg, Germany , respectively.

Information about the experimental design and statistical rationale for the different analyses performed in this work are provided within the respective subsections in the results.

Unless otherwise stated, boxes generally represent the median as a line surrounded by a box, which represents the percentiles of the data. Whiskers range within the percentiles.

Numbers of sample size, replicates, controls, and statistical tests were chosen according to published data with comparable methodology and generally accepted standards. No image processing, other than cropping, scaling and contrast adjustment, was applied. Quantification of Western blot signals was performed with Quantity One 4. For statistical analysis, paired two-sided t -tests were performed using OriginPro 9.

To minimize the effects of subjective bias, Western blot data were generated and analyzed by two different experimenters. Further information on research design is available in the Nature Research Reporting Summary linked to this article. Uncropped images of Western blots, sequence alignments, constructs used for cell transfection and bacterial transformation as well as bright field and fluorescence microscopic pictures are shown in Supplementary Figs.

Molecular mass markers and the outlines of cropping presented in the main figures are indicated. Manning, B. Cell , — Jiang, B. Natl Acad. USA 96 , — Rommel, C. Science , — Yang, G. Cell Rep. Laplante, M. Brunet, A. Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor.

Cell 96 , — This library served as tool to determine the mRNA frequencies for a number of hematopoietic marker proteins. Moreover, in a proteome analysis of cultured basophils we identified MBP and EPO as the two most prominent protein bands, suggesting a good correlation between protein and mRNA analyses of these cells.

The mixed phenotype observed for these cells strengthens the conclusion that eosinophils and basophils are closely linked during human hematopoietic development.

The dual phenotype also indicates that other cytokines than IL-3 or cell surface interactions are needed to obtain the full basophil specific phenotype in vivo. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist. Basophils have historically often been described as blood mast cells MCs since they both stain positive for basic dyes and contain histamine.

However, despite large similarities in humans they clearly represent two separate cell lineages. In contrast, MCs are much more common, are rarely found in peripheral blood and are essentially resident tissue cells [1]. They were given their name due to the fact that they stain with basic dyes like Toluidine Blue and Alcian blue. Basophils are primarily known for their central role in IgE-mediated allergies.

However, recently basophils have also attracted major interest as a potential key initiator for the induction of T-helper type 2 Th2 immune responses to invading pathogens. Supporting evidence for this Th2 priming function of the basophil comes from studies demonstrating that basophils express and release substantial amounts of IL-4, IL and thymic stromal lymphopoietin TSLP [3] , [4] , [5] , [6] , [7] , [8] , [9] , [10]. In favor of a major importance for the cytokines produced by basophils is the fact that the mouse MC protease mMCP -8 positive basophils identified during malaria infection produce up to 40 times more IL-4 than fully activated Th2 cells [5].

Basophils have also been identified as the most important source of IL-4 during the first hours after antigen challenge [7]. IL-4 and IL are both key cytokines in Th2 mediated immune responses and these are the only two cytokines known to induce B cells to switch to IgE [11]. A number of studies have recently also shown the importance of basophils in various inflammatory conditions.

They have been shown to play a pivotal role in the induction of eosinophil infiltration during the induction phase of atopic dermatitis [12]. They are also essential for IgG but not IgE mediated anaphylaxis in mice [13]. Interestingly, basophils have been shown to be of major importance for the memory response to ectoparasites, like ticks [14].

From numerous studies the general view has emerged that basophils and mast cells have distinct roles in allergy and asthma, as reviewed in [15] , and that basophils are involved in the defense against both endo- and ecto-parasites [16] , [17] , [18].

Although basophils have been known for more than years, very little is known about their granule content, especially for human basophils. It has been shown that human basophils store small amounts of the mast cell tryptase Blom and Hellman unpublished results and [19] , whereas human MCs store large amounts of proteases, belonging to both the tryptase and the chymase families [20] , [21].

However, no basophil specific human protein has yet been cloned and no homologue to mMCP-8 can be identified in the human genome [25] , [26]. Interestingly, the mMCP-8 gene including its regulatory elements has recently been used to specifically ablate basophils showing the specificity of this protein marker to murine basophils [14] , [16] , [27].

Two human basophils specific proteins have been identified basogranulin and a protein reacting against monoclonal 2D7 [28] , [29]. However, none of these have been characterized in more detail or cloned, in spite of massive efforts.

A major obstacle in the identification of new basophil-specific proteins has been the difficulty in obtaining sufficient numbers of pure basophils in combination with an almost complete absence of functional mRNA in peripheral blood basophils. The explanation for the extremely low mRNA levels is most likely that, like certain other leukocytes, i. The cloning of novel lineage-specific genes from other granulocytes have therefore almost exclusively come from the use of immortalized tumor cell lines [30] or in vitro -cultivated cells [31].

These cells are actively dividing and contain large amounts of mRNA. Both of these cell lines do however display characteristics of multiple lineages, suggesting that they are not optimal for studies of lineage-specific granule proteins. An alternative is therefore to use cultured basophil precursors. It is well established that IL-3, in human cord-blood or bone marrow cultures, promotes the expansion of a cell type closely resembling basophils as demonstrated by cell surface expression, staining properties and functional characteristics [35] , [36] , [37].

In order to obtain immature transcriptionally active basophils for an analysis of their transcriptome and to clone human basophil specific proteins a project was initiated to study conditions needed to obtain immature basophils in sufficient amounts to construct a high quality cDNA library. Such culture conditions were optimized and we managed to obtain preparations of more than 34 million immature basophils for mRNA purification and cDNA library construction [38].

CDc has also been found to be up-regulated upon activation of basophils as reviewed in [40]. A cDNA library from a preparation of these cells were produced and used as a starting material for a transcriptome analysis.

Upon sequence analysis of a panel of randomly picked clones from this library we found that eosinophil related transcripts were present in relatively high numbers, indicating that these cells have a mixed phenotype, displaying both basophil- and eosinophil-related characteristics [38].

We here present a more detailed characterization of this library using a more extensive sequence analysis of a larger number of randomly picked clones and a screening with specific probes. These experiments were performed to give a more detailed picture of the phenotype of IL-3 induced cord blood derived basophils cells as well as an evaluation of their usefulness in studying basophil biology.

Buffy coats were obtained from the University Hospital in Uppsala. The retrieved cells were then purified by a combination of negative and positive selection by magnetic-activated cell sorting MACS Miltenyi Biotec, Bergisch-Gladbach, Germany.

During negative selection a 2-step cocktail procedure was used. Antibody binding was performed on ice for 30 min with gentle shaking. The cells were then subjected at a positive selection step.

After informed consent and after approval from the local Ethical Review Board at the University Hospital, Uppsala, umbilical cord blood was collected from normal, full-term deliveries. The medium was changed after the first seven days. In particular, basophils are thought to be important in immune responses to parasites including tick and filarial worms. Basophils and mast cells have long been implicated in the pathogenesis of allergic disease as high levels of mediators common to both cell types are found in tissue locations relevant to allergic diseases.

Basophils are also a source of the major Th2-driving cytokine, IL-4 , early in immune responses. Basophils are rapidly recruited to the skin, lung or nose, following antigen challenge in humans, and are found in elevated numbers in asthma, allergic rhinitis, atopic dermatitis and nasal polyps. In these conditions recruited basophils participate in late phase reactions by the production and release of a number of mediators such as histamine , LTC4 and IL Thus basophils may fulfil pathological roles in both the onset and chronicity of allergic disease.

Recent studies in mice suggest that basophils may also regulate the behavior of T cells and mediate the magnitude of the secondary immune response. When activated, basophils degranulate to release histamine, proteoglycans e. They also secrete lipid mediators like leukotrienes, and several cytokines. Histamine and proteoglycans are pre-stored in the cell's granules while the other secreted substances are newly generated.

Each of these substances contributes to inflammation. Recent evidence suggests that basophils are an important source of the cytokine, interleukin-4, perhaps more important than T cells.