Phone staining and you may circulate cytometry.
Single-cell suspensions were prepared from different lymphoid organs and incubated for 10 min at 10 6 cells/20 ?l on ice in staining buffer (phosphate-buffered saline [PBS] containing 0.5% bovine serum albumin [BSA] and 0.01% NaNstep 3) with optimal amounts of fluorescein isothiocyanate-, phycoerythrin-, or biotin-conjugated antibodies. ): S7 (anti-CD43), B3B4 (anti-CD23), and Ly1 (anti-CD5). The following antibodies were prepared: RA3-6B2 (anti-B220), R33- (anti-IgM), 1.3-5 (anti-IgD), and Cfo-1 (anti-Thy1.2). Flow cytometric analysis was performed on a FACScan cytometer (Becton Dickinson & Co., Mountain View, Calif.).
Studies off B-telephone growth and you may upregulation out of activation indicators.
Splenic B tissue was in fact purified because of the exhaustion out of low-B cells for the MACs columns (Miltenyi Biotec, Bergisch Gladbach, Germany) with anti-CD43 antibody paired to help you magnetic beads (Miltenyi Biotec) while the explained in past times (17). New love off B structure try controlled by fluorescence-triggered phone sorter investigation, together with arrangements from B structure Date Asian Woman studiepoeng from 95% love were utilized. B structure had been sparked with goat anti-IgM antibody (2.5 ?g/ml) (Diaburg, Germany), anti-CD40 antibody (0.six ?g/ml) (Pharmingen), and you may IL-4 (twenty-five U/ml) (Genzyme Corp., Boston, Bulk.). The analysis of cell growth and you will upregulation away from activation indicators are did because the described before (3, 14).
Analysis off necessary protein phrase and you will tyrosine phosphorylation.
For the analysis of protein expression, cells were lysed in lysis buffer (10% glycerol, 1% Triton X-100, 20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 ?g of leupeptin per ml, 10 ?g of aprotinin per ml). The lysates equivalent to 5 ? 10 6 cells were loaded onto a sodium doide gel, and the separated proteins were electrotransferred to a Hybond nitrocellulose filter (Amersham) by semidry method. After being subjected to blocking with PBS–0.5% BSA–0.1% Tween 20, the filter was incubated first with a rabbit polyclonal antibody that recognizes the unique domain of Blk plus the SH3 and SH2 domains and then with a horseradish peroxidase-conjugated goat anti-rabbit IgG (Amersham) and developed with the enhanced chemiluminescence system (Amersham). RNA was analyzed by Northern blot analysis (20) using a blk cDNA probe (6). This probe (2,094 bp) contains the entire Blk coding sequence. For the analysis of tyrosine phosphorylation of whole-cell lysates and specific substrates downstream of Blk, purified B cells were suspended in RPMI supplemented with 2% fetal calf serum and stimulated with 20 ?g of F(ab?)2 fragment of goat anti-mouse IgM per ml for the indicated time (see Fig. ? Fig.3) 3 ) at 37°C. After centrifugation, cells were lysed in lysis buffer containing 1% Nonidet P-40. Whole-cell lysates corresponding to 5 ? 10 5 cells were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) (10% polyacrylamide). The rest of the lysates (representing 2.5 ? 10 6 cells) were incubated with either anti-Syk (a generous gift from C. A. Lowell), anti-phospholipase C-?2 (PLC?2) (Santa Cruz, Santa Cruz, Calif.), or anti-Grb2 (Transduction Laboratories) antibodies for 1 h and then with protein A-Sepharose (Pharmacia) for 30 min. Sepharose beads were washed three times with lysis buffer and subjected to SDS-PAGE. The proteins were transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, Mass.), incubated with PY99 antiphosphotyrosine antibody (Santa Cruz), and detected with the Supersignal System (Pierce, Rockford, Ill.). For the analysis of protein tyrosine phosphorylation, purified B cells were suspended in serum-free RPMI 1640 for 1 h and then stimulated for 15 min at 37°C with 15 ?g of goat anti-mouse IgM per ml at a density of 5 ? 10 7 cells/ml. The cells were pelleted by centrifugation and then lysed in lysis buffer containing 1% Nonidet P-40. The cell lysate was clarified by centrifugation for 10 min at 12,000 ? g. Aliquots of supernatants were incubated for 2 h with 20 ?g of bead-immobilized glutathione S-transferase–Blk SH2 domain fusion protein or 10 ?g of bead-immobilized antiphosphotyrosine monoclonal antibody 4G10. The beads were collected by centrifugation and washed four times with lysis buffer. The pellets were boiled in SDS-PAGE loading buffer, and the protein was fractionated by electrophoresis through an SDS–8% polyacrylamide gel. The protein was transferred to nitrocellulose. Phosphotyrosine-containing proteins were detected by immunoblotting with 4G10 antibody, and the membrane-bound antibody was detected by enhanced chemiluminescence.