Immunoblotting and qPCR Analysis of B Cell Activation

B cells were stimulated for 0-3 days, centrifuged, and suspended in Laemmli lysis buffer. Samples were separated by SDS-PAGE gel electrophoresis using a 4% stacking layer with either an 8% polyacrylamide gel for ATAD5 and β-actin, or a 15% polyacrylamide gel for PCNA and AID. Proteins were transferred to a polyvinylidene difluoride membrane (Biorad) and detected with rabbit anti-ATAD5 (12 (link)), mouse anti-β-actin (clone AC-15, Sigma-Aldrich), rabbit anti-PCNA (Abcam), or rabbit anti-AID (13 (link)) antibodies with cognate whole molecule anti-horseradish peroxidase (Sigma-Aldrich). Chemiluminescent signals (Pierce ECL Plus Substrate) were quantified in ImageJ software. For AID transcript levels, RNA was harvested after 3 d stimulation using an RNeasy Mini Kit (Qiagen). cDNA was reverse-transcribed using RNase H minus M-MLV point mutant (Promega) with oligo(dT) primers (Qiagen), followed by qPCR using iTaq SYBR Green Supermix with ROX (Biorad) with previously published primers (14 (link)).

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Zanotti K.J., Maul R.W., Castiblanco D.P., Yang W., Choi Y.J., Fox J.T., Myung K., Saribasak H, & Gearhart P.J. (2014). ATAD5 Deficiency Decreases B Cell Division and Igh Recombination. Journal of immunology (Baltimore, Md. : 1950), 194(1), 35-42.

Publication 2014

polyvinylidene difluoride membrane mouse anti-β-actin (clone AC-15, rabbit anti-PCNA RNeasy Mini Kit oligo(dT) primers iTaq SYBR Green Supermix with ROX

Actin Anti antibodies Atad5 B cells Cdna Clone Electrophoresis Horseradish peroxidase Laemmli buffer Membrane Mouse Oligo Pcna Polyacrylamide gel Polyvinylidene difluoride Primers Promega Proteins Rabbit Rnase h Rnase m Sds page Sybr green

Corresponding Organization :

Other organizations : MRC Laboratory of Molecular Biology, National Human Genome Research Institute, National Institutes of Health

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Variable analysis

independent variables

Duration of B cell stimulation (0-3 days)

dependent variables

Protein levels of ATAD5, β-actin, PCNA, and AID
AID transcript levels

control variables

Laemmli lysis buffer
SDS-PAGE gel electrophoresis
4% stacking layer and 8% or 15% polyacrylamide gel
Protein transfer to polyvinylidene difluoride membrane
Antibodies used for detection: rabbit anti-ATAD5, mouse anti-β-actin, rabbit anti-PCNA, rabbit anti-AID
Chemiluminescent signal quantification using ImageJ software
RNA harvesting and cDNA synthesis methods
QPCR using published primers

positive controls

None specified

negative controls

None specified

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