Quantifying Western blots: Pitfalls of densitometry

Gassmann, Max; Grenacher, Beat; Rohde, Bianca; Vogel, Johannes

doi:10.1002/elps.200800720

Cited by 355 publications

(278 citation statements)

References 14 publications

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“…ImageJ allowed us to measure the density profiles, peak heights, and peak intensity of the band of any expected molecular weight. Background correction was done using Rolling Ball Background Subtraction (rolling ball radius¼250; Gassmann et al, 2009). The contours of the rectangular boxes were determined arbitrarily using a width of approximately 30% of the lane and long enough to completely outline the bands of interest and the neighboring background.…”

Section: Western Blotsmentioning

confidence: 99%

Cdc42 GTPase and Rac1 GTPase act downstream of p120 catenin and require GTP exchange during gastrulation of zebrafish mesoderm

Hsu

Muerdter

Knickerbocker

et al. 2012

Developmental Dynamics

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Background: We investigated the roles of p120 catenin, Cdc42, Rac1, and RhoA GTPases in regulating migration of presomitic mesoderm cells in zebrafish embryos. p120 catenin has dual roles: It binds the intracellular and juxtamembrane region of cadherins to stabilize cadherin-mediated adhesion with the aid of RhoA GTPase, and it activates Cdc42 GTPase and Rac1 GTPase in the cytosol to initiate cell motility. Results: During gastrulation of zebrafish embryos, knockdown of the synthesis of zygotic p120 catenind1 mRNAs with a splice-site morpholino caused lateral widening and anterior-posterior shortening of the presomitic mesoderm and somites and a shortened anterior-posterior axis. These phenotypes indicate a cell-migration effect. Co-injection of low amounts of wild-type Cdc42 or wild-type Rac1 or dominant-negative RhoA mRNAs, but not constitutively-active Cdc42 mRNA, rescued these p120 catenin d1-depleted embryos. Conclusions: These downstream small GTPases require appropriate spatiotemporal stimulation or cycling of GTP to guide mesodermal cell migration. A delicate balance of Rho GTPases and p120 catenin underlies normal development. Developmental Dynamics 241:1545-1561, 2012. V C 2012 Wiley Periodicals Inc.Key words: p120 Catenin (CTNND1); ARVCF; Delta-catenin (CTNND2b); Cdc42 GTPase; Rac1 GTPase, RhoA GTPase; gastrulation; presomitic mesoderm; somites; zebrafish Key findings p120 catetin is required for extension of the dorsal axis and normal migration of the presomitic mesoderm. Cdc42 and Rac1 GTPases are downstream of p120 catenin d1 signaling and require exchange of GTP for GDP. Local stimulation of the exchange of GTP for GDP in Cdc42 and Rac GTPases mediates directional migration of the presomitic mesoderm. A balance of the amount of p120 catenin d1 and localized activation or turnover of Cdc42, Rac1, and Rho GTPase are required for normal zebrafish cell migration. Accepted 31 July 2012 Developmental DynamicsABBREVIATIONS Ab antibody ARVCF armadillo repeat gene deleted in velo-cardio-facial syndrome CA constitutively active Chr chromosome C(t) relative amount of RT-PCR product hpf hours post-fertilization DN dominant negative d1 splice-MO antisense morpholino oligonucleotide to the 12 th splice site of zebrafish p120 catenin d1 p120 catenin d1 (CTNND1) also called p120 catenin, Xenopus p120 catenin is a CTNND1 p120 catenin d2b (CTNND2b) also called Delta-catenin Rok1 Rho kinase1 RT reverse transcriptase minus-RT controls without reverse transcriptase qRT-PCR quantitative real-time PCR WT wild-type Xp120 catenin mRNA Xenopus p120 catenin d1 mRNA.Additional Supporting Information may be found in the online version of this article.

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Section: Western Blotsmentioning

confidence: 99%

Cdc42 GTPase and Rac1 GTPase act downstream of p120 catenin and require GTP exchange during gastrulation of zebrafish mesoderm

Hsu

Muerdter

Knickerbocker

et al. 2012

Developmental Dynamics

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“…Promising clones were then successively refined by two additional rounds of limiting dilution seeding and screening by Western blot. Quantification of Cul4B knockdown was performed using ImageJ (National Institutes of Health) as previously published (27). Nontarget control shRNA expressing RAW264.7 cells were maintained as a mixed population selected based on their resistance to 6 mg/ml puromycin.…”

Section: Immunoblottingmentioning

confidence: 99%

Cullin 4B Is Recruited to Tristetraprolin-Containing Messenger Ribonucleoproteins and Regulates TNF-α mRNA Polysome Loading

Pfeiffer

Brooks

2012

The Journal of Immunology

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TNF-α is a central mediator of inflammation and critical for host response to infection and injury. TNF-α biosynthesis is controlled by transcriptional and posttranscriptional mechanisms allowing for rapid, transient production. Tristetraprolin (TTP) is an AU-rich element binding protein that regulates the stability of the TNF-α mRNA. Using a screen to identify TTP-interacting proteins, we identified Cullin 4B (Cul4B), a scaffolding component of the Cullin ring finger ligase family of ubiquitin E3 ligases. Short hairpin RNA knockdown of Cul4B results in a significant reduction in TNF-α protein and mRNA in LPS-stimulated mouse macrophage RAW264.7 cells as well as a reduction in TTP protein. TNF-α message t1/2 was reduced from 69 to 33 min in LPS-stimulated cells. TNF-3′ untranslated region luciferase assays utilizing wild-type and mutant TTP-AA (S52A, S178A) indicate that TTP function is enhanced in Cul4B short hairpin RNA cells. Importantly, the fold induction of TNF-α mRNA polysome loading in response to LPS stimulation is reduced by Cul4B knockdown. Cul4B is present on the polysomes and colocalizes with TTP to exosomes and processing bodies, which are sites of mRNA decay. We conclude that Cul4B licenses the TTP-containing TNF-α messenger ribonucleoprotein for loading onto polysomes, and reduction of Cul4B expression shunts the messenger ribonucleoproteins into the degradative pathway.

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“…Image densitometry. Western blot quantification was meticulously performed using a procedure we developed based on the recommendations of Gassmann et al 38 Specifically, all quantified immunoblots were revealed using the same type of films (X-OMAT Blue XB, cat. 1776699, PerkinElmer, Waltham, MA, USA) and carefully exposed to avoid saturation.…”

Section: Discussionmentioning

confidence: 99%

Thromboxane A2 modulates cisplatin-induced apoptosis through a Siva1-dependent mechanism

Iorio-Morin

Germain

et al. 2012

Cell Death Differ

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Thromboxane A 2 (TXA 2 ) is an important lipid mediator whose function in apoptosis is the subject of conflicting reports. Here, a yeast two-hybrid screen for proteins that interact with the C-terminus of the TXA 2 receptor (TP) identified Siva1 as a new TP-interacting protein. Contradictory evidence suggests pro-and anti-apoptotic roles for Siva1. We show that a cisplatin treatment induces TXA 2 synthesis in HeLa cells. We demonstrate that endogenous TP stimulation promotes cisplatin-induced apoptosis of HeLa cells and that such modulation requires the expression of Siva1, as evidenced by inhibiting its endogenous expression using siRNAs. We reveal that, upon stimulation of TP, degradation of Siva1 is impeded, resulting in an accumulation of the protein, which translocates from the nucleus to the cytosol. Translocation of Siva1 correlates with its reduced interaction with Mdm2 (an inhibitor of p53 signalling), as well as with its increased interaction with TRAF2 and XIAP (known to enhance pro-apoptotic signalling). Our data provide a model that reconciles the pro-and anti-apoptotic roles that were reported for Siva1 and identify a new mechanism for promoting apoptosis by G protein-coupled receptors. Our findings may have implications in the use of cyclo-oxygenase inhibitors during cisplatin chemotherapy and might provide a target to reduce cisplatin toxicity on non-cancerous tissues.

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Quantifying Western blots: Pitfalls of densitometry

Cited by 355 publications

References 14 publications

Cdc42 GTPase and Rac1 GTPase act downstream of p120 catenin and require GTP exchange during gastrulation of zebrafish mesoderm

Cdc42 GTPase and Rac1 GTPase act downstream of p120 catenin and require GTP exchange during gastrulation of zebrafish mesoderm

Cullin 4B Is Recruited to Tristetraprolin-Containing Messenger Ribonucleoproteins and Regulates TNF-α mRNA Polysome Loading

Thromboxane A2 modulates cisplatin-induced apoptosis through a Siva1-dependent mechanism

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