Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Fiala, Gina J.; Schamel, Wolfgang W. A.; Blumenthal, Britta

doi:10.3791/2164

Cited by 57 publications

(58 citation statements)

References 12 publications

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“…BN-PAGE assay was performed according to a method described by Fiala et al (55) and Wittig et al (56). Details are described in SI Appendix, Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope

Tsai

Chuang

Tsai

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

115

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The sigma-1 receptor (Sig-1R) chaperone at the endoplasmic reticulum (ER) plays important roles in cellular regulation. Here we found a new function of Sig-1R, in that it translocates from the ER to the nuclear envelope (NE) to recruit chromatin-remodeling molecules and regulate the gene transcription thereof. Sig-1Rs mainly reside at the ER-mitochondrion interface. However, on stimulation by agonists such as cocaine, Sig-1Rs translocate from ER to the NE, where Sig-1Rs bind NE protein emerin and recruit chromatin-remodeling molecules, including lamin A/C, barrier-to-autointegration factor (BAF), and histone deacetylase (HDAC), to form a complex with the gene repressor specific protein 3 (Sp3). Knockdown of Sig-1Rs attenuates the complex formation. Cocaine was found to suppress the gene expression of monoamine oxidase B (MAOB) in the brain of wild-type but not Sig-1R knockout mouse. A single dose of cocaine (20 mg/kg) in rats suppresses the level of MAOB at nuclear accumbens without affecting the level of dopamine transporter. Daily injections of cocaine in rats caused behavioral sensitization. Withdrawal from cocaine in cocaine-sensitized rats induced an apparent time-dependent rebound of the MAOB protein level to about 200% over control on day 14 after withdrawal. Treatment of cocaine-withdrawn rats with the MAOB inhibitor deprenyl completely alleviated the behavioral sensitization to cocaine. Our results demonstrate a role of Sig-1R in transcriptional regulation and suggest cocaine may work through this newly discovered genomic action to achieve its addictive action. Results also suggest the MAOB inhibitor deprenyl as a therapeutic agent to block certain actions of cocaine during withdrawal.T he endoplasmic reticulum (ER) plays important roles in cellular functions, including synthesis of proteins and regulation of Ca 2+ signaling between the ER and plasma membrane (1) and between the ER and mitochondria (2-4). However, because the ER interacts with other organelles in the cell, other functions related to the ER remain to be uncovered.The sigma-1 receptor (Sig-1R) (5-8) is an ER chaperone molecule that resides at the ER-mitochondrion interface referred to as the mitochondria-associated ER membrane (MAM), where the Sig-1R ensures proper ER-mitochondrion Ca 2+ signaling for cellular survival (3, 9), as well as sustains the activity of an ER stress sensor IRE1 at the MAM (10). The Sig-1R can translocate from the MAM to plasma membrane of the cell to regulate ion channels and receptors on the plasma membrane (8,(11)(12)(13)(14). Cocaine is a Sig-1R agonist (3) that causes the dissociation of Sig-1R from its cognate binding partner BiP (3,8), and consequently the translocation of Sig-1Rs to the plasma membrane, where Sig-1Rs interact with voltage-gated potassium channel subfamily A member 2 (Kv1.2) to shape the neuronal and behavioral responses to cocaine (15).Cocaine also causes the translocation of Sig-1Rs from the ER to the nucleus (16), where Sig-1Rs are shown to be present at the nuclear envelope (NE) (17). H...

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“…BN-PAGE assay was performed according to a method described by Fiala et al (55) and Wittig et al (56). Details are described in SI Appendix, Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope

Tsai

Chuang

Tsai

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

115

View full text Add to dashboard Cite

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“…The protein solutions were desalted by ultrafiltration with Amicon Ultra 15 mL 30 KD Tubes (Millipore, Billerica, MA). The sizes of the native protein and of the monomer were assessed by blue-native polyacrylamide gel electrophoresis (Fiala et al, 2011) and sodium dodecyl-sulfate-polyacrylamide gel electrophoresis, respectively.…”

Section: Purification Of His-tagged Fdh Proteinsmentioning

confidence: 99%

A Formate Dehydrogenase Confers Tolerance to Aluminum and Low pH

et al. 2016

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Formate dehydrogenase (FDH) is involved in various higher plant abiotic stress responses. Here, we investigated the role of rice bean (Vigna umbellata) VuFDH in Al and low pH (H + ) tolerance. Screening of various potential substrates for the VuFDH protein demonstrated that it functions as a formate dehydrogenase. Quantitative reverse transcription-PCR and histochemical analysis showed that the expression of VuFDH is induced in rice bean root tips by Al or H + stresses. Fluorescence microscopic observation of VuFDH-GFP in transgenic Arabidopsis plants indicated that VuFDH is localized in the mitochondria. Accumulation of formate is induced by Al and H + stress in rice bean root tips, and exogenous application of formate increases internal formate content that results in the inhibition of root elongation and induction of VuFDH expression, suggesting that formate accumulation is involved in both H + -and Al-induced root growth inhibition. Over-expression of VuFDH in tobacco (Nicotiana tabacum) results in decreased sensitivity to Al and H + stress due to less production of formate in the transgenic tobacco lines under Al and H + stresses. Moreover, NtMATE and NtALS3 expression showed no changes versus wild type in these over-expression lines, suggesting that herein known Al-resistant mechanisms are not involved. Thus, the increased Al tolerance of VuFDH over-expression lines is likely attributable to their decreased Al-induced formate production. Taken together, our findings advance understanding of higher plant Al toxicity mechanisms, and suggest a possible new route toward the improvement of plant performance in acidic soils, where Al toxicity and H + stress coexist.

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“…Cells were incubated in lysis buffer (20 mM Bis-tris, pH 7.0, 500 mM ε-aminocaproic acid, 20 mM NaCl, 0.2 mM EDTA, 0.5% Triton X-100, 10% glycerol, and protease inhibitor cocktail) and then dialyzed against lysis buffer containing 0.1% Triton X-100 overnight (41). Samples were loaded onto a 4%-15% gradient gel and resolved for 3.5 hours at 150 V. The gel was transferred to a PVDF membrane (30 V for 8 hours) using Tris-glycine buffer (pH 8.3).…”

Section: Author Contributionsmentioning

confidence: 99%

SHARPIN-mediated regulation of protein arginine methyltransferase 5 controls melanoma growth

Tamiya¹,

Kim²,

Klymenko³

et al. 2017

Journal of Clinical Investigation

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Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Cited by 57 publications

References 12 publications

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope

Sigma-1 receptor mediates cocaine-induced transcriptional regulation by recruiting chromatin-remodeling factors at the nuclear envelope

A Formate Dehydrogenase Confers Tolerance to Aluminum and Low pH

SHARPIN-mediated regulation of protein arginine methyltransferase 5 controls melanoma growth

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