Reversible Ponceau staining as a loading control alternative to actin in Western blots

Romero‐Calvo, Isabel; Ocón, Borja; Martínez-Moya, Patricia; Suárez, Marı́a Dolores; Zarzuelo, Antonio; Martínez‐Augustin, Olga

doi:10.1016/j.ab.2010.02.036

Cited by 668 publications

(474 citation statements)

References 5 publications

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“…All membranes were stained with Ponceau S, and digital images were acquired for the control of protein loading by densitometric analysis (36,37). Membrane was blocked in PBS-Tween containing 5% nonfat dry milk at room temperature, followed by incubation with COX-1 goat polyclonal (1:500; Santa Cruz), COX-2 mouse polyclonal (1:1,000; Cayman), and PKCe rabbit polyclonal antibody (1:1,000; Santa Cruz) overnight at 4°C, rinsed six The asterisk (*) indicates a response significantly greater (P < 0.05, unpaired t test) than that of other groups.…”

Section: Odn-asmentioning

confidence: 99%

Peripheral inflammatory hyperalgesia depends on the COX increase in the dorsal root ganglion

Araldi

Ferrari

Lotufo

et al. 2013

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

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It is well established that dorsal root ganglion (DRG) cells synthesize prostaglandin. However, the role that prostaglandin plays in the inflammatory hyperalgesia of peripheral tissue has not been established. Recently, we have successfully established a technique to inject drugs (3 μL) directly into the L5-DRG of rats, allowing in vivo identification of the role that DRG cell-derived COX-1 and COX-2 play in the development of inflammatory hyperalgesia of peripheral tissue. IL-1β (0.5 pg) or carrageenan (100 ng) was administered in the L5-peripheral field of rat hindpaw and mechanical hyperalgesia was evaluated after 3 h. Administration of a nonselective COX inhibitor (indomethacin), selective COX-1 (valeryl salicylate), or selective COX-2 (SC-236) inhibitors into the L5-DRG prevented the hyperalgesia induced by IL-1β. Similarly, oligodeoxynucleotide-antisense against COX-1 or COX-2, but not oligodeoxynucleotide-mismatch, decreased their respective expressions in the L5-DRG and prevented the hyperalgesia induced by IL-1β in the hindpaw. Immunofluorescence analysis demonstrated that the amount of COX-1 and COX-2, constitutively expressed in TRPV-1 + cells of the DRG, significantly increased after carrageenan or IL-1β administration. In addition, indomethacin administered into the L5-DRG prevented the increase of PKCe expression in DRG membrane cells induced by carrageenan. Finally, the administration of EP1/EP2 (7.5 ng) or EP4 (10 μg) receptor antagonists into L5-DRG prevented the hyperalgesia induced by IL-1β in the hindpaw. In conclusion, the results of this study suggest that the inflammatory hyperalgesia in peripheral tissue depends on activation of COX-1 and COX-2 in C-fibers, which contribute to the induction and maintenance of sensitization of primary sensory neurons.D uring tissue injury, prostaglandin-E 2 (PGE 2 ) is produced by the activation of the enzyme cyclooxygenase (COX) to play an important role in inflammatory hyperalgesia. PGE 2 sensitizes peripheral nociceptors through the activation of PGE 2 receptors (EP) (1). This sensitization, characterized by a reduction of nociceptive threshold and by an increase in peripheral afferent neuron responsiveness, is the main feature of inflammatory hyperalgesia in the peripheral tissue. The widespread use of nonsteroidal antiinflammatory drugs to control inflammatory hyperalgesia exemplifies the relevance of PGE 2 for the development of inflammatory hyperalgesia. These drugs decrease peripheral inflammatory hyperalgesia by inhibiting COX and, therefore, by preventing the synthesis of PGE 2 (2, 3).The COX enzyme is expressed in two major isoforms, COX-1 and COX-2, with different biological functions (4), although both play a role in the inflammatory hyperalgesia (5, 6). The isoform of COX constitutively expressed in dorsal root ganglion (DRG) is still unclear. Although COX-1 mRNA and, to a lesser extent, COX-2 mRNA have been detected in DRG cultures (7) under basal conditions, immunohistochemical studies have shown that DRG cells express only COX-1, but not C...

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Section: Odn-asmentioning

confidence: 99%

Peripheral inflammatory hyperalgesia depends on the COX increase in the dorsal root ganglion

Araldi

Ferrari

Lotufo

et al. 2013

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

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“…The mean optical densities of immunoreactive bands were measured by computer-assisted densitometry (GS-800 Calibrated Densitometer; Bio-Rad), and compared to a linear concentration curve as previously described (Churn et al, 1992). In the SPAR and PSD-95 experiments, data were normalized to whole-lane optical densities obtained from the same Ponceau S-stained blots (Romero-Calvo et al, 2010).…”

Section: Western Blot Analysismentioning

confidence: 99%

Mechanisms of Dendritic Spine Remodeling in a Rat Model of Traumatic Brain Injury

Campbell

Low

Kurz

et al. 2012

Journal of Neurotrauma

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Traumatic brain injury (TBI), a leading cause of death and disability in the United States, causes potentially preventable damage in part through the dysregulation of neural calcium levels. Calcium dysregulation could affect the activity of the calcium-sensitive phosphatase calcineurin (CaN), with serious implications for neural function. The present study used both an in vitro enzymatic assay and Western blot analyses to characterize the effects of lateral fluid percussion injury on CaN activity and CaN-dependent signaling in the rat forebrain. TBI resulted in an acute alteration of CaN phosphatase activity and long-lasting alterations of its downstream effector, cofilin, an actin-depolymerizing protein. These changes occurred bilaterally in the neocortex and hippocampus, appeared to persist for hours after injury, and coincided with synapse degeneration, as suggested by a loss of the excitatory post-synaptic protein PSD-95. Interestingly, the effect of TBI on cofilin in some brain regions was blocked by a single bolus of the CaN inhibitor FK506, given 1 h post-TBI. Overall, these findings suggest a loss of synapse stability in both hemispheres of the laterally-injured brain, and offer evidence for region-specific, CaN-dependent mechanisms.

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“…Thus, even wild type HEK 293 cells may express a very low background level of FPR1. Ponceau S staining after stripping of the membranes was used to verify equal protein loading (34) (Fig. 2D) as the cells had intrinsic differences in the amount of ␤-actin attached to the membranes.…”

Section: Fpr1 Is Expressed By Human Lens Epithelial Cells-fpr1mentioning

confidence: 99%

The Leukocyte Chemotactic Receptor FPR1 Is Functionally Expressed on Human Lens Epithelial Cells

Schneider

Weaver

Gaur

et al. 2012

Journal of Biological Chemistry

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Reversible Ponceau staining as a loading control alternative to actin in Western blots

Cited by 668 publications

References 5 publications

Peripheral inflammatory hyperalgesia depends on the COX increase in the dorsal root ganglion

Peripheral inflammatory hyperalgesia depends on the COX increase in the dorsal root ganglion

Mechanisms of Dendritic Spine Remodeling in a Rat Model of Traumatic Brain Injury

The Leukocyte Chemotactic Receptor FPR1 Is Functionally Expressed on Human Lens Epithelial Cells

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