Anti phospho shp1

Whole cell lysates were electrophoresed on 10% sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were incubated with primary antibodies overnight at 4°C followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies. The following antibodies were used: anti-CREB, anti-Phospho-CREB (Ser133), anti-SHP-1, anti-Phospho-SHP1 (Tyr564), anti-SHP-2, anti-Phospho-SHP2 (Tyr580), anti-ITGB1, anti-ITGB3, p-CaMK2, CaMK2 (Cell Signaling Technology), anti-ANGPTL2 (R&D Systems), anti-CaMK1 (Abcam), anti-p-CaMK4 (Abmart), anti-CaMK4 (Genscript), anti-Flag (Sigma), anti-GAPDH, and anti-β-actin (Calbiochem). Anti-LILRB2-PE (eBioscience) and isotype control of IgG-PE were used to detect the expression of LILRB2 on the different lung cancer cell lines and analyzed by flow cytometry.

Purified memory B cells were equilibrated in complete medium at 37°C for 10 min, and prewarmed particulates coated with biotinylated CpG and biotinylated anti BCR or biotinylated anti-BCR were added at a ratio of 5,000 particulates per B cell. Soluble CpG was added at a final concentration of 1 µg/ml for the indicated time. Cells were lysed in 1% NP-40 buffer and analyzed by SDS-PAGE followed by immunoblotting with indicated antibodies. Anti–phospho-Lyn (Y507), anti–phospho-ERK, anti–phospho-Akt (Ser473), anti–phospho-Syk, anti–phospho-PLCϒ2, anti–phospho-CD19, anti–phospho-SHP-1, and anti-ERK were all from Cell Signaling Technology. The density of the bands was quantified by densitometry, corrected for background, normalized to the density of the actin band in the same sample, and made relative to the unstimulated zero time point for each condition.

For surface expression analysis, cells were prepared as outlined above and 1 × 10⁶ were aliquoted into FACS tubes. Cells were pelleted by centrifugation and each tube was resuspended in flow buffer [1% (w/v) BSA, 4 mmol/L EDTA, and 0.15 mmol/L NaN₃ in PBS] containing anti-CD19 and anti-CD5 antibodies for CLL cell detection. Antibodies against specific key markers and surface phosphatases were then used in various combinations. Cells were incubated on ice in the dark for 15 minutes prior to washing with flow buffer. Cells were resuspended in flow buffer and analyzed using a Canto cytometer. Data were analyzed using FlowJo.
For intracellular phosphatases, lysates were analyzed by immunoblotting with the following antibodies: anti-phospho-SHIP1, anti-phospho-SHP1, anti-phospho-LYN (Y507; all Cell Signaling Technology), anti-SHP1, anti-phospho-LYN (Y396; both Abcam), anti-SHIP1, anti-LYN (both Insight Biotechnology), anti-phospho-PTPN22 (R&D Systems), and anti-GAPDH (Thermo Fisher Scientific). Primary antibodies were probed using species-specific HRP-conjugated secondary antibodies (Dako) and viewed using a Chemidoc imager (Bio-Rad).