Methods and Protocols

All standard methods and protocols used for studies described within this report are listed. Detailed recipes for preparation of solutions are also available and specified within each protocol. Additional methods or modifications to standard protocols are described below the table.

Preparative 2-D Gel Electrophoresis

WEHI-231 cells (1.5 x 10⁷) were grown in medium containing fetal bovine serum. Primary splenic B cells (1.5 x 10⁸) were isolated as described above (PP00000001) and incubated for 1 hour in serum-free Dulbecco's Modified Eagle's Medium (DMEM). Cells were collected by centrifugation and washed once with phosphate buffered saline. The cell pellets were lysed with IEF lysis buffer (8M urea, 4% CHAPS, 20mM Tris-HCl, pH 9, 5 ml/ml Pefabloc, 1X Complete Mini Protease Cocktail [Roche], 1mM sodium orthovanadate, 1 mM okadaic acid) at a ratio of 50 ml of buffer per 1 x 10⁷ cells. The lysates were then sonicated 2 times for 3 seconds and ultracentrifuged for 1 hour at 22^oC at 70,000 x g. The protein in the supernatant was precipitated with TCA/acetone and resuspended in rehydration buffer (8M urea, 4% CHAPS, 1% immobilized pH gradient [IPG] buffer, 13 mM DTT, 5 ml/ml Pefabloc, 1X Complete Mini Protease Cocktail [Roche], 1mM sodium orthovanadate, 1mM okadaic acid). The proteins were then separated by 2-D electrophoresis (protocols available on the AfCS Web site). The standard protocols were followed except the glass plates were treated with Bind Silane (Amersham Biosciences) before gel casting to keep the gel attached to the plate during spot picking. Preparative 2-D gels were stained with either Colloidal Blue (Invitrogen) or SYPRO Ruby (Bio-Rad) according to the manufacturer's protocols. The gels were then imaged in a 2-D Master Imager fluorescence scanner (Amersham Biosciences).

Mass Spectrometry

The Colloidal Blue- or SYPRO Ruby-stained LSP1 protein spots were excised and digested according to the AfCS protocol (PP00000156). The MS and MS/MS spectra were acquired by oMALDI-QqTOF mass spectrometry (PP00000157). The MS and MS/MS spectra were used to search the NCBI non-redundant mouse protein database using Knexus software (Genomic Solutions, Inc.). Nanoelectrospray precursor ion scanning and tandem mass spectrometry (MS/MS) (PP00000158) were used to identify phosphorylation sites in LSP1. The phosphorylation sites were identified by manual analysis of the fragmentation patterns of individual phosphopeptides.

Analytical 2-D Gel Electrophoresis

WEHI-231 cells (1.1 x 10⁷) were incubated in phosphate-free Supplemented Iscove's Modified Dulbecco's Medium (SIMDM), and primary B cells (2 x 10⁷) were incubated in phosphate-free DMEM, both containing 1 mCi/ml of ³²P inorganic phosphate, for 3 hours at 37^oC in 5% CO2. Cells were then treated with anti-IgM, IL-4 or PMA for 10 minutes according to AfCS protocols. Cells were collected by centrifugation.

For fluorescent labeling, the cell pellet was lysed with TTX-1 IEF lysis buffer (50mM Tris-HCl, pH 7.6, 300 mM NaCl, 0.5% Triton X-100, 1X complete protease inhibitor cocktail [Roche], 1 mM sodium orthovanadate, 1 mM okadaic acid) at a ratio of 10 m l of buffer per 1x10⁶ cells. The lysate was incubated on ice for 30 minutes with occasional vortexing. The sample was then centrifuged for 15 minutes at 12,000 x g at 4^oC. The proteins were labeled with Cy3 or Cy5 dyes* (Amersham Biosciences) according to the AfCS protocol, TCA precipitated, and resuspended in 2-D rehydration buffer 1 (AfCS protocol PS00000481) for 2-D electrophoresis.

For samples that were not fluorescently labeled, the cell pellet was lysed with TriPure at a ratio of 10 m l of buffer per 1x10⁶ cells, followed by protein extraction according to the AfCS protocol (PP00000154). The protein pellets were resuspended in 2-D rehydration buffer 1 for 2-D electrophoresis.

* We used Cy3 and Cy5 saturation dyes from Amersham Biosciences, available only to customers with a technology access agreement with Amersham. Amersham Biosciences recommends Cy3 minimal dye (catalog no. RPK0273) and Cy5 minimal dye (catalog no. RPK0275) as commercially available substitutes for the saturation dyes. These substitutes were not tested by the AfCS; therefore, we cannot predict if the substitutes will produce identical results to those detailed in this report.