Immunoprecipitation and Western Blot Analysis

Total cellular proteins were extracted as described (Sabbir et al., 2010 (link)). The cell lysate was pre-cleared and immunoprecipitated using anti-Dlc1 and anti-Myh9 antibodies as described (Sabbir et al., 2012 (link)). The western blots were immunoblotted with anti-GFP (ab-290), anti-Myh9 (Sc-98978, Santa Cruz Biotechnology (SCBT) Dallas, TX; ARP48072_P050, Aviva Systems Biology, San Diego, CA), anti-phospho-Myh9-Ser-1943 (CST-5026, New England Biolabs, Ipswich, MA), anti-Plectin (sc-33649, SCBT) and anti-Spectrin, antibodies (Sc-32931,SCBT) and visualized. Immuno-blots were scanned using a Storm 840 PhosphorImager scanner and quantified by densitometry using ImageQuant software (version 1.2; both from Molecular Dynamics, Sunnyvale, CA).

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Sabbir M.G., Dillon R, & Mowat M.R. (2016). Dlc1 interaction with non-muscle myosin heavy chain II-A (Myh9) and Rac1 activation. Biology Open, 5(4), 452-460.

Publication 2016

ab-290 ImageQuant software

Anti antibodies Antibodies Cell Densitometry Dlc1 Molecular dynamics Plectin Proteins Spectrin Western blots

Corresponding Organization :

Other organizations : Research Institute in Oncology and Hematology, CancerCare Manitoba, University of Manitoba

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Variable analysis

independent variables

Anti-Dlc1 antibody
Anti-Myh9 antibody

dependent variables

Total cellular proteins
Phospho-Myh9-Ser-1943
Plectin
Spectrin

control variables

Cell lysate pre-clearance

controls

Positive control: Not specified
Negative control: Not specified

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