Co-immunoprecipitation was performed using 800 μl HTR8-CM or 400 μl pooled BCM 200 μl or 100 μl of lysis buffer respectively and 1 μg of Ago1 antibody (Cell signalling technologies), Ago 2 antibody (Cell signalling technologies) or control IgG (Dako). Following Incubation at 4 °C overnight, the immune complexes were pulled down with protein A/G magnetic beads (Thermo Scientific) and serially washed with 0.5% TBS/Tween, followed by TBS and distilled H2O. 500 μl of TriReagent was added to each sample and RNA extracted as per standard protocol described above. Method adapted from (Arroyo et al., 2011 (link)).
Control igg
Manufactured by Agilent Technologies
Sourced in United Kingdom
Control IgG is a laboratory reagent used in immunoassays and other immunological techniques. It serves as a control sample to help ensure the accuracy and reliability of the test results.
Automatically generated - may contain errors
2 protocols using control igg
1
Ago1/Ago2 Co-immunoprecipitation from HTR8-CM and BCM
2
Maspin Expression in Cell Lines
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Cell Lines, Immunological Markers, Protein, Peptides MCF-7 and DU145 cell lines with differing maspin expression generated and maintained as before (Ravenhill et al. 2010) . Cell lines were authenticated by the DNA Diagnostics Centre (London, UK). Primary aortic smooth muscle cells (referred to as VSMC) were cultured as detailed previously (Bass et al. 2009) . HT1080 were obtained from ATCC and maintained in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% (v/v) foetal calf serum (FCS). All cell culture reagents, Alexa Fluor 568 labelled phalloidin and secondary antibodies were from Life Technologies (Paisley, UK). Maspin peptides (Ravenhill et al. 2010 ) and recombinant maspin (Bass et al. 2009 ) as detailed previously. Commercially available mouse monoclonal antibodies were used to detect maspin (BD Biosciences, Oxford, UK). Control IgG was from Dako (Ely, UK).
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