Immunoblotting for Protein Expression

Immunoblotting for DOCK8 protein and β-actin was performed as previously described (Zhang et al., 2009 (link)). For analysis of other proteins, cells were lysed in 2% SDS (50 mM Tris HCl, pH 6.8, 10% glycerol) or 1% NP-40 (1 mM EDTA, 50 mM Hepes, and 150 mM NaCl) containing complete protease inhibitors (Roche). Protein lysates were separated on NuPAGE Novex 12% or 4–12% gradient Bis-Tris gels using MOPS SDS or MES running buffers (Life Technologies), followed by wet or semi-dry transfer. Membranes were blocked with 5% blocking grade nonfat milk (Bio-Rad Laboratories) or bløk-CH (Millipore). Antibodies were to caspase-9 (BD); caspase-8 (clone C15; gift from L. Zheng, NIAID, NIH, Bethesda, MD); and PAK1, PAK2, and WASP (Santa Cruz Biotechnology, Inc.).

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Zhang Q., Dove C.G., Hor J.L., Murdock H.M., Strauss-Albee D.M., Garcia J.A., Mandl J.N., Grodick R.A., Jing H., Chandler-Brown D.B., Lenardo T.E., Crawford G., Matthews H.F., Freeman A.F., Cornall R.J., Germain R.N., Mueller S.N, & Su H.C. (2014). DOCK8 regulates lymphocyte shape integrity for skin antiviral immunity. The Journal of Experimental Medicine, 211(13), 2549-2566.

Publication 2014

complete protease inhibitors blocking grade nonfat milk caspase-9

Actin Antibodies Bis tris Buffers Caspase 8 Caspase 9 Cells Clone Dock8 Edta Gels Glycerol Hepes Membranes Milk Mops Nacl Np 40 Pak1 Protease inhibitors Protein Tris Wasp

Corresponding Organization : ARC Centre of Excellence in Advanced Molecular Imaging

Other organizations : MRC Human Immunology Unit, University of Oxford

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Variable analysis

independent variables

Immunoblotting method (as previously described in Zhang et al., 2009)
Cell lysis method (2% SDS or 1% NP-40 with protease inhibitors)
Protein separation method (NuPAGE Novex gels with MOPS SDS or MES running buffers)

dependent variables

Levels of DOCK8 protein
Levels of β-actin protein
Levels of other unspecified proteins

control variables

Blocking reagents (5% blocking grade nonfat milk or bløk-CH)
Antibodies used (caspase-9, caspase-8, PAK1, PAK2, WASP)

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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