A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background

Haan, Claude; Behrmann, Iris

doi:10.1016/j.jim.2006.07.027

Cited by 225 publications

(166 citation statements)

References 15 publications

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“…Proteins were transferred from SDS-PAGE and blue native gels onto a PVDF membrane (EMD Millipore) via semi-dry Western blotting. Immunosignals were detected by enhanced chemiluminescence (97) and visualized on x-ray films (Medix XBU) or via the LAS3000 image reader (FujiFilm). …”

Section: Methodsmentioning

confidence: 99%

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

Schuler¹,

Bartolomeo

Mårtensson

et al. 2016

Journal of Biological Chemistry

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Two protein translocases transport precursor proteins into or across the inner mitochondrial membrane. The presequence translocase (TIM23 complex) sorts precursor proteins with a cleavable presequence either into the matrix or into the inner membrane. The carrier translocase (TIM22 complex) inserts multispanning proteins into the inner membrane. Both protein import pathways depend on the presence of a membrane potential, which is generated by the activity of the respiratory chain. The non-bilayer-forming phospholipids cardiolipin and phosphatidylethanolamine are required for the activity of the respiratory chain and therefore to maintain the membrane potential for protein import. Depletion of cardiolipin further affects the stability of the TIM23 complex. The role of bilayer-forming phospholipids like phosphatidylcholine (PC) in protein transport into the inner membrane and the matrix is unknown. Here, we report that import of presequence-containing precursors and carrier proteins is impaired in PC-deficient mitochondria. Surprisingly, depletion of PC does not affect stability and activity of respiratory supercomplexes, and the membrane potential is maintained. Instead, the dynamic TIM23 complex is destabilized when the PC levels are reduced, whereas the TIM22 complex remains intact. Our analysis further revealed that initial precursor binding to the TIM23 complex is impaired in PC-deficient mitochondria. We conclude that reduced PC levels differentially affect the TIM22 and TIM23 complexes in mitochondrial protein transport.Mitochondria fulfill essential functions for the survival of the cell like energy conversion to produce ATP, synthesis of amino acids, lipids, and heme, as well as the generation of iron-sulfur clusters. They contain about 1000 proteins in yeast and 1500 proteins in humans (1, 2). More than 99% of the mitochondrial proteins are synthesized as precursors on cytosolic ribosomes. Mitochondria contain a sophisticated system of protein translocases to import precursor proteins (3-9). The translocase of the outer membrane (TOM 3 complex) forms the general entry gate for most precursor proteins. After passage of the TOM channel, distinct protein translocases sort the preproteins into the different subcompartments: the outer and inner membrane as well as the two aqueous compartments, the matrix and intermembrane space.The majority of mitochondrial proteins are sorted into the inner membrane and the matrix. Two inner membrane-bound protein complexes mediate protein import. The presequence translocase (also termed TIM23 complex) transports precursor proteins with a cleavable presequence into the inner membrane and the matrix, whereas the carrier translocase (also termed TIM22 complex) inserts proteins with multiple transmembrane segments into the inner membrane (3-9). The membrane potential across the inner membrane provides the driving force for both protein import pathways and is generated by the activity of the respiratory chain. Presequence-containing preproteins are directly transferred from the ...

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Section: Methodsmentioning

confidence: 99%

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

Schuler¹,

Bartolomeo

Mårtensson

et al. 2016

Journal of Biological Chemistry

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“…After three washes in TBS-0.05% Tween, the membranes were incubated with the appropriate HRP-conjugated secondary Abs for 2 h at room temperature. After three further washes in TBS-0.05% Tween, the membranes were incubated with HRP substrate solution (22) and exposed to light-sensitive film for various times. Rac1-GTP levels were assessed according to the manufacturers' protocol (Millipore).…”

Section: Sds-page and Western Blottingmentioning

confidence: 99%

L-Plastin Regulates Polarization and Migration in Chemokine-Stimulated Human T Lymphocytes

Freeley

O’Dowd

Paul

et al. 2012

The Journal of Immunology

105

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Chemokines such as SDF-1α play a crucial role in orchestrating T lymphocyte polarity and migration via polymerization and reorganization of the F-actin cytoskeleton, but the role of actin-associated proteins in this process is not well characterized. In this study, we have investigated a role for L-plastin, a leukocyte-specific F-actin–bundling protein, in SDF-1α–stimulated human T lymphocyte polarization and migration. We found that L-plastin colocalized with F-actin at the leading edge of SDF-1α–stimulated T lymphocytes and was also phosphorylated at Ser5, a site that when phosphorylated regulates the ability of L-plastin to bundle F-actin. L-plastin phosphorylation was sensitive to pharmacological inhibitors of protein kinase C (PKC), and several PKC isoforms colocalized with L-plastin at the leading edge of SDF-1α–stimulated lymphocytes. However, PKC ζ, an established regulator of cell polarity, was the only isoform that regulated L-plastin phosphorylation. Knockdown of L-plastin expression with small interfering RNAs demonstrated that this protein regulated the localization of F-actin at the leading edge of chemokine-stimulated cells and was also required for polarization, lamellipodia formation, and chemotaxis. Knockdown of L-plastin expression also impaired the Rac1 activation cycle and Akt phosphorylation in response to SDF-1α stimulation. Furthermore, L-plastin also regulated SDF-1α–mediated lymphocyte migration on the integrin ligand ICAM-1 by influencing velocity and persistence, but in a manner that was independent of LFA-1 integrin activation or adhesion. This study, therefore, demonstrates an important role for L-plastin and the signaling pathways that regulate its phosphorylation in response to chemokines and adds L-plastin to a growing list of proteins implicated in T lymphocyte polarity and migration.

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“…Signals were detected using an ECL solution containing 2.5 mM Luminol (SigmaAldrich: 123072), 2.6 mM hydrogenperoxide (SigmaAldrich: H1009), 100mM Tris-HCL/ pH8.8 (SigmaAldrich: T-1503) and 0.2 mM para-coumaric acid (SigmaAldrich: C9008). 60 Prior to reprobing, the blots were stripped in 2%SDS (Carl Roth: CN30.3), 100 mM b-mercaptoethanol (Carl Roth: 4227.3) in 62.5 mM Tris-HCl (pH6.7/ for 30 min at 70 C).…”

Section: Cloning and Expression Vectorsmentioning

confidence: 99%

The oncogenic FIP1L1-PDGFRαfusion protein displays skewed signaling properties compared to its wild-type PDGFRαcounterpart

et al. 2015

Self Cite

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ABSTRACT. Aberrant activation of oncogenic kinases is frequently observed in human cancers, but the underlying mechanism and resulting effects on global signaling are incompletely understood. Here, we demonstrate that the oncogenic FIP1L1-PDGFRa kinase exhibits a significantly different signaling pattern compared to its PDGFRa wild type counterpart. Interestingly, the activation of primarily membrane-based signal transduction processes (such as PI3-kinase-and MAP-kinase-pathways) is remarkably shifted toward a prominent activation of STAT factors. This diverging signaling pattern compared to classical PDGF-receptor signaling is partially coupled to the aberrant cytoplasmic localization of the oncogene, since membrane targeting of FIP1L1-PDGFRa restores activation of MAPK-and PI3K-pathways. In stark contrast to the classical cytokine-induced STAT activation process, STAT activation by FIP1L1-PDGFRa does neither require Janus kinase activity nor Src kinase activity. Furthermore, we investigated the mechanism of STAT5 activation via FIP1L1-PDGFRa in more detail and found that STAT5 activation does not involve an SH2-domain-mediated binding mechanism. We thus demonstrate that STAT5 activation occurs via a non-canonical activation mechanism in which STAT5 may be subject to a direct phosphorylation by FIP1L1-PDGFRa.

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A cost effective non-commercial ECL-solution for Western blot detections yielding strong signals and low background

Cited by 225 publications

References 15 publications

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

L-Plastin Regulates Polarization and Migration in Chemokine-Stimulated Human T Lymphocytes

The oncogenic FIP1L1-PDGFRαfusion protein displays skewed signaling properties compared to its wild-type PDGFRαcounterpart

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