Clean western blot signals from immunoprecipitated samples

Lal, Ashish; Haynes, Susan R.; Gorospe, Myriam

doi:10.1016/j.mcp.2005.06.007

Cited by 45 publications

(37 citation statements)

References 7 publications

(10 reference statements)

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“…Immunoprecipitation Assays-The immunoprecipitation assays were prepared as described previously (51). For immunoprecipitation, protein A-Sepharose beads (Sigma) were coated with the appropriate antibodies against ATF-3 (Santa Cruz Biotechnology), BRG1 (Santa Cruz Biotechnology), or nonspecific IgG as a control.…”

Section: Methodsmentioning

confidence: 99%

Recruitment of SWI/SNF Complex Is Required for Transcriptional Activation of the SLC11A1 Gene during Macrophage Differentiation of HL-60 Cells

Thuraisingam

Marino

et al. 2011

Journal of Biological Chemistry

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The solute carrier family 11 member 1 (SLC11A1) gene is strictly regulated and exclusively expressed in myeloid lineage cells. However, little is known about the transcriptional regulation of the SLC11A1 gene during myeloid development. In this study, we used HL-60 cells as a model to investigate the regulatory elements/factors involved in the transactivation of the SLC11A1 gene during phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation of HL-60 cells. Promoter deletion analysis showed that a 7-base AP-1-like element (TGACTCT) was critical for the responsiveness of the SLC11A1 promoter to PMA. Stimulation by PMA induced the binding of ATF-3 and the recruitment of two components of the SWI/SNF complex, BRG1 and ␤-actin, to this element in an ATF-3-dependent manner. RNAi-mediated depletion of ATF-3 or BRG1 markedly decreased SLC11A1 gene expression and its promoter activity induced by PMA. Luciferase reporter experiments demonstrated that ATF-3 cooperated with BRG1 and ␤-actin to activate the SLC11A1 promoter. Furthermore, we showed that PMA can induce the proximal (GT/AC) n repeat sequence to convert to the Z-DNA structure in the SLC11A1 gene promoter, and depletion of BRG1 resulted in a significant decrease of Z-DNA formation. Our results demonstrated that recruitment of the SWI/SNF complex initiated Z-DNA formation and subsequently helped to transactivate the SLC11A1 gene.The solute carrier family 11 member 1 (SLC11A1) gene, also known as Ity/Lsh/BCG or natural resistance-associated macrophage 1 (NRAMP1) gene, is associated with host resistance to infection. In mice, mutations in the Slc11a1 gene, whether naturally occurring or experimentally induced, cause susceptibility to infection with unrelated intracellular pathogens such as Salmonella, Leishmania, and Mycobacteria (1-5). The multiple pleiotropic effects of the SLC11A1 gene on macrophage activation, including regulation of the chemokine KC and cytokines (e.g. TNF␣), as well as induction of nitric oxide (NO) release, MHC class II molecule expression, and oxidative burst (6, 7), display its potential importance in autoimmune and infectious diseases. In humans, polymorphic variants of the SLC11A1 gene are associated with susceptibility to infectious diseases such as tuberculosis, leprosy, and HIV infection, as well as autoimmune diseases such as rheumatoid arthritis, sarcoidosis, diabetes, and Crohn disease (6,8). Furthermore, the polymorphisms of the SLC11A1 gene have been linked with esophageal cancer risk (9).In humans, the SLC11A1 gene is located on chromosome 2q35 and has 15 exons spanning about 14 kb. The gene encodes a transmembrane protein exclusively expressed in the myeloid lineage as follows: monocytes, macrophages, polymorphonuclear neutrophils, and dendritic cells (10, 11). SLC11A1 gene expression is strictly regulated during myeloid development, and SLC11A1 protein expression parallels with its mRNA level (12), suggesting that SLC11A1 expression may be controlled primarily at the level of transcription. The human promyelocy...

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Section: Methodsmentioning

confidence: 99%

Recruitment of SWI/SNF Complex Is Required for Transcriptional Activation of the SLC11A1 Gene during Macrophage Differentiation of HL-60 Cells

Thuraisingam

Marino

et al. 2011

Journal of Biological Chemistry

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“…Immunoprecipitated proteins were recovered by boiling the beads in 2% SDS and 12% Suc for 50 s. After centrifugation, the supernatant was complemented to 0.1 M DTT and 0.1 M Na 2 CO 3 and analyzed by SDS-PAGE. Protein gel blots of immunoprecipitated samples were detected using horseradish peroxidase-conjugated protein A instead of horseradish peroxidase-conjugated secondary antibodies to decrease the background (Lal et al, 2005).…”

Section: Bn-page and Immunoprecipitationsmentioning

confidence: 99%

Thylakoid FtsH Protease Contributes to Photosystem II and Cytochromeb 6 fRemodeling inChlamydomonas reinhardtiiunder Stress Conditions

et al. 2014

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FtsH is the major thylakoid membrane protease found in organisms performing oxygenic photosynthesis. Here, we show that FtsH from Chlamydomonas reinhardtii forms heterooligomers comprising two subunits, FtsH1 and FtsH2. We characterized this protease using FtsH mutants that we identified through a genetic suppressor approach that restored phototrophic growth of mutants originally defective for cytochrome b 6 f accumulation. We thus extended the spectrum of FtsH substrates in the thylakoid membranes beyond photosystem II, showing the susceptibility of cytochrome b 6 f complexes (and proteins involved in the c i heme binding pathway to cytochrome b 6 ) to FtsH. We then show how FtsH is involved in the response of C. reinhardtii to macronutrient stress. Upon phosphorus starvation, photosynthesis inactivation results from an FtsH-sensitive photoinhibition process. In contrast, we identified an FtsH-dependent loss of photosystem II and cytochrome b 6 f complexes in darkness upon sulfur deprivation. The D1 fragmentation pattern observed in the latter condition was similar to that observed in photoinhibitory conditions, which points to a similar degradation pathway in these two widely different environmental conditions. Our experiments thus provide extensive evidence that FtsH plays a major role in the quality control of thylakoid membrane proteins and in the response of C. reinhardtii to light and macronutrient stress.

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“…For detection of coimmunoprecipitated b-arrestin-1, horseradish peroxidase-conjugated protein A (Pierce, Rockford, IL) was used as secondary antibody. 33 …”

Section: Immunoblotting and Immunoprecipitationmentioning

confidence: 99%

β-Arrestin-1 Drives Endothelin-1–Mediated Podocyte Activation and Sustains Renal Injury

Buelli

Rosanò²,

Gagliardini

et al. 2014

Journal of the American Society of Nephrology

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Activation of endothelin-A receptor (ET A R) by endothelin-1 (ET-1) drives epithelial-to-mesenchymal transition in ovarian tumor cells through b-arrestin signaling. Here, we investigated whether this pathogenetic pathway could affect podocyte phenotype in proliferative glomerular disorders. In cultured mouse podocytes, ET-1 caused loss of the podocyte differentiation marker synaptopodin and acquisition of the mesenchymal marker a-smooth muscle actin. ET-1 promoted podocyte migration via ET A R activation and increased b-arrestin-1 expression. Activated ET A R recruited b-arrestin-1 to form a trimeric complex with Src leading to epithelial growth factor receptor (EGFR) transactivation and b-catenin phosphorylation, which promoted gene transcription of Snail. Increased Snail expression fostered ET-1-induced migration as confirmed by Snail knockdown experiments. Silencing of b-arrestin-1 prevented podocyte phenotypic changes and motility and inhibited ET A R-driven signaling. In vitro findings were confirmed in doxorubicin (Adriamycin)-induced nephropathy. Mice receiving Adriamycin developed renal injury with loss of podocytes and hyperplastic lesion formation; b-arrestin-1 expression increased in visceral podocytes and in podocytes entrapped in pseudo-crescents. Administration of the selective ET A R antagonist sitaxsentan prevented podocyte loss, formation of the hyperplastic lesions, and normalized expression of glomerular b-arrestin-1 and Snail. Increased b-arrestin-1 levels in podocytes retrieved from crescents of patients with proliferative glomerulopathies confirmed the translational relevance of these findings and suggest the therapeutic potential of ET A R antagonism for a group of diseases still needing a specific treatment.

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Clean western blot signals from immunoprecipitated samples

Cited by 45 publications

References 7 publications

Recruitment of SWI/SNF Complex Is Required for Transcriptional Activation of the SLC11A1 Gene during Macrophage Differentiation of HL-60 Cells

Recruitment of SWI/SNF Complex Is Required for Transcriptional Activation of the SLC11A1 Gene during Macrophage Differentiation of HL-60 Cells

Thylakoid FtsH Protease Contributes to Photosystem II and Cytochromeb 6 fRemodeling inChlamydomonas reinhardtiiunder Stress Conditions

β-Arrestin-1 Drives Endothelin-1–Mediated Podocyte Activation and Sustains Renal Injury

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