Tubulin or Not Tubulin: Heading Toward Total Protein Staining as Loading Control in Western Blots

Moritz, Christian P.

doi:10.1002/pmic.201600189

Cited by 128 publications

(101 citation statements)

References 110 publications

(226 reference statements)

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“…Antibodies were diluted in Wes Antibody Diluent to the final working concentrations: p65, 1:50 (Cell Signaling, D14E12; Beverly, MA); phospho‐Ser139 H2A.X, 1:50 (Cell Signaling, 20E3); HT7, 1:1,000 (Pierce/Invitrogen); NeuN, 1:50 (Millipore, MAB377; Temecula, CA, USA): GFAP 1:200 (Cell Signaling, D1F4Q); synaptophysin, 1:50 (Cell Signaling, D35E4); see Supporting Information Table S1 for complete antibody information. Protein quantification was performed by normalizing to total protein concentration (Li & Shen, 2013; Moritz, 2017; Supporting Information Figure S8). …”

Section: Methodsmentioning

confidence: 99%

Tau protein aggregation is associated with cellular senescence in the brain

et al. 2018

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Tau protein accumulation is the most common pathology among degenerative brain diseases, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), traumatic brain injury (TBI), and over twenty others. Tau‐containing neurofibrillary tangle (NFT) accumulation is the closest correlate with cognitive decline and cell loss (Arriagada, Growdon, Hedley‐Whyte, & Hyman, 1992), yet mechanisms mediating tau toxicity are poorly understood. NFT formation does not induce apoptosis (de Calignon, Spires‐Jones, Pitstick, Carlson, & Hyman, 2009), which suggests that secondary mechanisms are driving toxicity. Transcriptomic analyses of NFT‐containing neurons microdissected from postmortem AD brain revealed an expression profile consistent with cellular senescence. This complex stress response induces aberrant cell cycle activity, adaptations to maintain survival, cellular remodeling, and metabolic dysfunction. Using four AD transgenic mouse models, we found that NFTs, but not Aβ plaques, display a senescence‐like phenotype. Cdkn2a transcript level, a hallmark measure of senescence, directly correlated with brain atrophy and NFT burden in mice. This relationship extended to postmortem brain tissue from humans with PSP to indicate a phenomenon common to tau toxicity. Tau transgenic mice with late‐stage pathology were treated with senolytics to remove senescent cells. Despite the advanced age and disease progression, MRI brain imaging and histopathological analyses indicated a reduction in total NFT density, neuron loss, and ventricular enlargement. Collectively, these findings indicate a strong association between the presence of NFTs and cellular senescence in the brain, which contributes to neurodegeneration. Given the prevalence of tau protein deposition among neurodegenerative diseases, these findings have broad implications for understanding, and potentially treating, dozens of brain diseases.

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Section: Methodsmentioning

confidence: 99%

Tau protein aggregation is associated with cellular senescence in the brain

et al. 2018

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“…Membranes were incubated with Ponceau S (PS) stain and the total optical density of each lane was quantified to verify equal loading. This approach eliminates potential impacts of treatment on commonly used single-protein loading controls (Aldridge, Podrebarac, Greenough, & Weiler, 2008;Gardan-Salmon, Dixon, Lonergan, & Selsby, 2011;Moritz, 2017). Membranes were washed in 1X tris buffered saline with 0.2% Tween20 (1X TBST) and blocked with 5% milk in 1X TBST for 1 hr.…”

Section: Western Blotmentioning

confidence: 99%

PGC‐1α overexpression increases transcription factor EB nuclear localization and lysosome abundance in dystrophin‐deficient skeletal muscle

et al. 2020

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Duchenne muscular dystrophy (DMD) is caused by the absence of functional dystrophin protein and results in progressive muscle wasting. Dystrophin deficiency leads to a host of dysfunctional cellular processes including impaired autophagy. Autophagic dysfunction appears to be due, at least in part, to decreased lysosomal abundance mediated by decreased nuclear localization of transcription factor EB (TFEB), a transcription factor responsible for lysosomal biogenesis. PGC‐1α overexpression decreased disease severity in dystrophin‐deficient skeletal muscle and increased PGC‐1α has been linked to TFEB activation in healthy muscle. The purpose of this study was to determine the extent to which PGC‐1α overexpression increased nuclear TFEB localization, increased lysosome abundance, and increased autophagosome degradation. We hypothesized that overexpression of PGC‐1α would drive TFEB nuclear translocation, increase lysosome biogenesis, and improve autophagosome degradation. To address this hypothesis, we delivered PGC‐1α via adeno‐associated virus (AAV) vector injected into the right limb of 3‐week‐old mdx mice and the contralateral limbs received a sham injection. At 6 weeks of age, this approach increased PGC‐1α transcript by 60‐fold and increased TFEB nuclear localization in gastrocnemii from PGC‐1α treated limbs by twofold compared to contralateral controls. Furthermore, lamp2, a marker of lysosome abundance, was significantly elevated in muscles from limbs overexpressing PGC‐1α. Lastly, increased LC3II and similar p62 in PGC‐1α overexpressing‐limbs compared to contralateral limbs are supportive of increased degradation of autophagosomes. These data provide mechanistic insight into PGC‐1α‐mediated benefits to dystrophin‐deficient muscle, such that increased TFEB nuclear localization in dystrophin‐deficient muscle leads to increased lysosome biogenesis and autophagy.

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“…A second limitation may be the use of a whole‐well Ponceau S stain as the normalizing method in lieu of the traditional use of a housekeeping marker. The use of total protein staining as a loading control over housekeeping proteins has several advantages, such as ease of use, reductions in cost, and reliability over different experimental outcomes . The working linear range of a Ponceau S stain when used as a loading control with kidney and liver tissue is between 10 and 140 µg per lane, but the intensity of the stain may not be sensitive to small changes in protein loading which may occur during Western blotting, and it may overvalue the expected protein amount, albeit with less variance than glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) or β‐actin …”

Section: Discussionmentioning

confidence: 99%

“…The use of total protein staining as a loading control over housekeeping proteins has several advantages, such as ease of use, reductions in cost, and reliability over different experimental outcomes. 51 The working linear range of a Ponceau S stain when used as a loading control with kidney and liver tissue is between 10 and 140 μg 20 per lane, but the intensity of the stain may not be sensitive to small changes in protein loading which may occur during Western blotting, 52 and it may overvalue the expected protein amount, albeit with less variance than glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-actin. 53 Skeletal muscle is largely composed of proteins of the contractile machinery and these proteins are targeted for rapid degradation in the first 2 weeks of unloading or paralysis.…”

Section: Discussionmentioning

confidence: 99%