A new background subtraction method for Western blot densitometry band quantification through image analysis software

Gallo-Oller, Gabriel; Ordóñez, Raquel; Dotor, Javier

doi:10.1016/j.jim.2018.03.004

Cited by 142 publications

(92 citation statements)

References 13 publications

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“…Antiactin (sc-1615; Santa Cruz, Dallas, TX, USA) or antivinculin antibody (#13901; Cell Signaling) served as control. Densitometric analyses comparing phosphorylated to non-phosphorylated proteins were performed by using Image J software and normalized to actin 38 .…”

Section: Analyses Of Blood and Liver Tissue Samplesmentioning

confidence: 99%

TNF-Receptor-1 inhibition reduces liver steatosis, hepatocellular injury and fibrosis in NAFLD mice

et al. 2020

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Non-alcoholic fatty liver disease (NAFLD) shows an increasing prevalence and is associated with the development of liver fibrosis and cirrhosis as the major risk factors of liver-related mortality in this disease. The therapeutic possibilities are limited and restricted to life style intervention, since specific drugs for NAFLD are unavailable so far. TNFα has been implicated as a major pathogenic driver of NAFLD. TNFα-mediated liver injury occurs mainly via TNF-receptor-1 (TNFR1) signaling, whereas TNFR2 mediates protective pathways. In this study, we analyzed the therapeutic effects of a novel antibody, which selectively inhibits TNFR1 while retaining protective TNFR2 signaling in a high-fat diet (HFD) mouse model of NAFLD. Mice were fed with HFD for 32 weeks and treated with anti-TNFR1-antibody or controlantibody for the last 8 weeks. We then investigated the mechanisms of TNFR1 inhibition on liver steatosis, inflammatory liver injury, insulin resistance and fibrosis. Compared to control-antibody treatment, TNFR1 inhibition significantly reduced liver steatosis and triglyceride content, which was accompanied by reduced expression and activation of the transcription factor SREBP1 and downstream target genes of lipogenesis. Furthermore, inhibition of TNFR1 resulted in reduced activation of the MAP kinase MKK7 and its downstream target JNK, which was associated with significant improvement of insulin resistance. Apoptotic liver injury, NAFLD activity and alanine aminotransferase (ALT) levels, as well as liver fibrosis significantly decreased by anti-TNFR1 compared to control-antibody treatment. Thus, our results suggest selective TNFR1 inhibition as a promising approach for NAFLD treatment.

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Section: Analyses Of Blood and Liver Tissue Samplesmentioning

confidence: 99%

TNF-Receptor-1 inhibition reduces liver steatosis, hepatocellular injury and fibrosis in NAFLD mice

et al. 2020

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“…The protein samples were separated by via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The immunoblots were blocked with 5% nonfat milk and subsequently incubated with the primary antibodies (1 : 1000) at 4 C overnight, followed by incubation with peroxidase conjugated second antibodies (1 : 5000) at room temperature for 2 h. 19 The protein bands were measured and b-actin was used as an internal standard of process control. The blot band densitometry was analyzed using the ImageJ soware.…”

Section: Western Blottingmentioning

confidence: 99%

Anti-inflammatory potency of Locusta migratoria manilensis cyclopeptides in mast cells and macrophages

Liu

Wang

et al. 2019

RSC Adv.

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“…High resolution pictures of gels were open in the ImageJ software. Using the area selection tool, a horizontal line was traced to cut pick that were then analyzed by their density, which was compared to density of WT strain for the bands corresponding to OmpF(Gallo-Oller et al, 2018).For extraction of RNA, overnight cultures were diluted 100 times and cultured in fresh LB or LB + CTET as appropriate, until reaching OD 600 0.5. RNA was extracted using the Trizol method according to the provider instructions.…”

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confidence: 99%

Inactivation of Glutamine Synthetase-Coding Gene glnA Increases Susceptibility to Quinolones Through Increasing Outer Membrane Protein F in Salmonella enterica Serovar Typhi

et al. 2020

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Ciprofloxacin is the choice treatment for infections caused by Salmonella Typhi, however, reduced susceptibility to ciprofloxacin has been reported for this pathogen. Considering the decreased approbation of new antimicrobials and the crisis of resistance, one strategy to combat this problem is to find new targets that enhances the antimicrobial activity for approved antimicrobials. In search of mutants with increased susceptibility to ciprofloxacin; 3,216 EZ-Tn5 transposon mutants of S. Typhi were screened. S. Typhi zxx::EZ-Tn5 mutants susceptible to ciprofloxacin were confirmed by agar diffusion and MIC assays. The genes carrying EZ-Tn5 transposon insertions were sequenced. Null mutants of interrupted genes, as well as inducible genetic constructs, were produced using site-directed mutagenesis, to corroborate phenotypes. SDS-PAGE and Real-time PCR were used to evaluate the expression of proteins and genes, respectively. Five mutants with increased ciprofloxacin susceptibility were found in the screening. The first confirmed mutant was the glutamine synthetase-coding gene glnA. Analysis of outer membrane proteins revealed increased OmpF, a channel for the influx of ciprofloxacin and nalidixic acid, in the glnA mutant. Expression of ompF increased four times in the glnA null mutant compared to WT strain. To understand the relationship between the expression of glnA and ompF, a strain with the glnA gene under control of the tetracycline-inducible P tet promoter was created, to modulate glnA expression. Induction of glnA decreased expression of ompF, at the same time that reduced susceptibility to ciprofloxacin. Expression of sRNA MicF, a negative regulator of OmpF was reduced to one-fourth in the glnA mutant, compared to WT strain. In addition, expression of glnL and glnG genes (encoding the two-component system NtrC/B that may positively regulate OmpF) were increased in the glnA mutant. Further Frontiers in Microbiology | www.frontiersin.org 1 March 2020 | Volume 11 | Article 428Millanao et al.glnA Inactivation Increases Quinolones Susceptibility studies indicate that deletion of glnG decreases susceptibility to CIP, while deletion of micF gene increases susceptibility CIP. Our findings indicate that glnA inactivation promotes ompF expression, that translates into increased OmpF protein, facilitating the entry of ciprofloxacin, thus increasing susceptibility to ciprofloxacin through 2 possible mechanisms.

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A new background subtraction method for Western blot densitometry band quantification through image analysis software

Cited by 142 publications

References 13 publications

TNF-Receptor-1 inhibition reduces liver steatosis, hepatocellular injury and fibrosis in NAFLD mice

TNF-Receptor-1 inhibition reduces liver steatosis, hepatocellular injury and fibrosis in NAFLD mice

Anti-inflammatory potency of Locusta migratoria manilensis cyclopeptides in mast cells and macrophages

Inactivation of Glutamine Synthetase-Coding Gene glnA Increases Susceptibility to Quinolones Through Increasing Outer Membrane Protein F in Salmonella enterica Serovar Typhi

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