Computational protocol: Engagement of Components of DNA-Break Repair Complex and NFκB in Hsp70A1A Transcription Upregulation by Heat Shock

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Protocol publication

[…] HeLa cells grown in 6 cm plates to ∼80% confluency were transfected with the FLAG-p65 and his6-HSF1 constructs. The cells were trypsinized 24 h post transfection to grow on the coverslip. Next day after subjecting to heat shock at 42°C/30 min (HS) or not (-HS) with or without 45 min recovery, coverslips were washed twice with PBS, fixed through treatment with 3.7% formaldehyde solution for 20 min. Following washing off the fixing solution with PBS, 0.2% titron X-100 was applied for 20 min (to enhance membrane permeabilization of cells). After blocking with 3% BSA, coverslips were incubated with anti-FLAG (1:250) or anti-his6 (1:250) antibody, washed, incubated with secondary antibody anti-mouse Alexa Flour 595 (1:100) or anti-rabbit TRITC (1:100), or anti-mouse Cyn5 (1:500) followed by incubation with DAPI (2 μg/ml) in the dark. After washing off the unbound materials with PBS, coverslips were mounted in DPX viewed and documented using a laser scanning confocal microscope (Leica TCS SP8, Leica Microsystems India Pvt. Ltd) using Leica Application Suite LAS X software. [...] The densitometric analyses of the bands visualised by ethidium bromide staining in semi-quantitative RT-PCR experiments were performed by the ImageJ (NIH) and ImageLab (BioRad) softwares. Statistical significance was analysed by one way anova with Turkey’s post test or Student’s t test by Graphpad Prism 5.0 software. A p value of less than 0.05 was considered statistically significant. […]

Pipeline specifications

Software tools LAS X, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Chemicals Poly Adenosine Diphosphate Ribose