High-Throughput Evaluation of Metabolic Activities Using Reverse Phase Protein Array Technology

Lu, Hsin‐Yi; Xiong, Jian; Perera, Dimuthu; Rajapakshe, Kimal I.; Wang, Xuan; Costello, Myra; Holloway, Kimberly; Ramos, Carlos Alberto do Nascimento; Grimm, Sandra L.; Wulfkuhle, Julia; Coarfa, Cristian; Edwards, Dean P.; Zhu, Michael X.; Huang, Shixia

doi:10.1201/9780367823269-13

Cited by 2 publications

(3 citation statements)

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“…Reverse phase protein array assays for antibodies to proteins or phosphorylated proteins in different functional pathways were carried out as described previously (Coarfa et al, 2021; Lu H.-Y., 2021; Wang et al, 2022). Specifically, protein lysates were prepared from cultured cells with modified Tissue Protein Extraction Reagent (TPER) (Life Technologies Corporation, Carlsbad, CA) and a cocktail of protease and phosphatase inhibitors (Roche, Pleasanton, CA) (Lu et.…”

Section: Methodsmentioning

confidence: 99%

“…The images were analyzed with GenePix Pro 7.0 (Molecular Devices, Silicon Valley, CA). Total fluorescence signal intensities of each spot were obtained after subtraction of the local background signal for each slide and were then normalized for variation in total protein, background and non-specific labeling using a group-based normalization method as described (Lu H.-Y., 2021). For each spot on the array, the-background-subtracted foreground signal intensity was subtracted by the corresponding signal intensity of the negative control slide (omission of primary antibody) and then normalized to the corresponding signal intensity of total protein for that spot.…”

Section: Methodsmentioning

confidence: 99%

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SOD1is a synthetic lethal target inPPM1D-mutant leukemia cells

Zhang,

Hsu,

Braekeleer

et al. 2023

Preprint

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The DNA damage response is critical for maintaining genome integrity and is commonly disrupted in the development of cancer. PPM1D (protein phosphatase, Mg2+/Mn2+ dependent 1D) is a master negative regulator of the response; gain-of-function mutations and amplifications of PPM1D are found across several human cancers making it a relevant pharmacologic target. Here, we used CRISPR/Cas9 screening to identify synthetic-lethal dependencies of PPM1D, uncovering superoxide dismutase-1 (SOD1) as a potential target for PPM1D-mutant cells. We revealed a dysregulated redox landscape characterized by elevated levels of reactive oxygen species and a compromised response to oxidative stress in PPM1D-mutant cells. Moreover, we observed marked genomic instability in mutant cells, which is exacerbated upon inhibition of SOD1. Altogether, our results demonstrate the protective role of SOD1 against oxidative stress and DNA damage in PPM1D-mutant leukemia cells and highlight a new potential therapeutic strategy against PPM1D-mutant cancers.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

SOD1is a synthetic lethal target inPPM1D-mutant leukemia cells

Zhang,

Hsu,

Braekeleer

et al. 2023

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…RPPA assays were carried out as described previously ( 79 , 80 ). Specifically, protein lysates were prepared from dissected cerebellar vermis samples with modified Tissue Protein Extraction Reagent (TPER) (Life Technologies Corporation, Carlsbad, CA) and a cocktail of protease and phosphatase inhibitors (Roche, Pleasanton, CA) ( 80 ). Lysates were diluted to 0.5 mg/ml in SDS sample buffer and denatured on the same day.…”

Section: Methodsmentioning

confidence: 99%

Activation of TrkB-Akt signaling rescues deficits in a mouse model of SCA6

et al. 2022

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Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease resulting in motor coordination deficits and cerebellar pathology. Expression of brain-derived neurotrophic factor (BDNF) is reduced in postmortem tissue from SCA6 patients. Here, we show that levels of cerebellar BDNF and its receptor, tropomyosin receptor kinase B (TrkB), are reduced at an early disease stage in a mouse model of SCA6 (SCA684Q/84Q). One month of exercise elevated cerebellar BDNF expression and improved ataxia and cerebellar Purkinje cell firing rate deficits. A TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF), likewise improved motor coordination and Purkinje cell firing rate and elevated downstream Akt signaling. Prolonged 7,8-DHF administration persistently improved ataxia when treatment commenced near disease onset but was ineffective when treatment was started late. These data suggest that 7,8-DHF, which is orally bioavailable and crosses the blood-brain barrier, is a promising therapeutic for SCA6 and argue for the importance of early intervention for SCA6.

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High-Throughput Evaluation of Metabolic Activities Using Reverse Phase Protein Array Technology

Cited by 2 publications

References 1 publication

SOD1is a synthetic lethal target inPPM1D-mutant leukemia cells

SOD1is a synthetic lethal target inPPM1D-mutant leukemia cells

Activation of TrkB-Akt signaling rescues deficits in a mouse model of SCA6

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