Anti phospho shp2

Cells were lyzed with RadioImmunoPrecipitation Assay (RIPA) buffer (Thermo Fisher Scientific, Villebon sur Yvette, France). Cell lysates were clarified by centrifugation at 14000 × g at 4°C for 15 min. For western blotting, proteins were separated by SDS-PAGE gels and transferred to a nitrocellulose membrane. Then, membranes were blocked with Tris buffered saline (TBS) (0.02 M Tris-HCl, 0.137 M NaCl, pH 7.4) containing 0.1% Tween (TBS-T) and 5% non-fat dry milk at room temperature during 1 hour and incubated overnight at 4°C with the following primary antibodies: anti-phospho-JAK2 (Santa Cruz Biotechnology), anti-GAPDH, anti-DDR1, anti-phospho-SHP2, anti-SHP2, anti-JAK2, anti-phospho-ERK1/2, anti-ERK1/2, anti-p21^CIP1 (Cell signaling Technology, Saint Quentin Yvelines, France), anti-DDR2 (R&D systems, Lille, France), anti-RAGE (GeneTex, Irvine, CA). Membranes were washed with TBS-T and incubated with the corresponding peroxidase conjugated secondary antibody at room temperature for 1 hour. Chemiluminescent detection was realized by using an ECL Prime Kit (GE Healthcare, Orsay, France).

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.