Odyssey clx quantitative ir fluorescent detection system

Ptpn11^fl/fl MEFs expressing CRE‐ER^Tam, Swiss 3T3 fibroblasts, or HEK293T cells were seeded on 6‐well plates (MEFs and 3T3 cells, 2 × 10⁵/well; HEK293T cells, 5 × 10⁵/well), followed by serum starvation (MEFs and 3T3 cells, no FBS; HEK293T cells, no FBS or 0.1% FBS) for 16 h. Starved cells were then stimulated with PDGF‐BB (50 ng·mL⁻¹, final concentration) or EGF (1 ng·mL⁻¹ or 50 ng·mL⁻¹, final concentration), as indicated. For inhibitor treatment, cells were pre‐incubated for 3 h (NSC‐87877) or 30 min (other SHP2 inhibitors) in media (1 mL/well) containing the indicated concentrations of each compound with 0.5% DMSO or 0.5% DMSO alone, followed by addition of 10 μL of medium containing 5 μg·mL⁻¹ PDGF‐BB (50 ng·mL⁻¹, final concentration). Cells were lysed in SDS lysis buffer (50 mm Tris/HCl pH 7.5, 100 mm NaCl, 1 mm EDTA, 1% SDS, 2 mm Na₃VO₄). Lysates were resolved by SDS/PAGE, followed by transfer to Immobilon‐FL PVDF membranes (Millipore). Membranes were blocked in 1% BSA/TBS containing 0.1% Tween‐20 for 30 min and treated with primary antibodies in blocking buffer for 1 h, followed by treatment with IRDye‐conjugated secondary antibodies (LI‐COR). Images were obtained using an ODYSSEY CLx quantitative IR fluorescent detection system (LI‐COR) with Image Studio software Ver. 5.2 (LI‐COR).

Cells were lysed in SDS lysis buffer (50 mM Tris-HCl pH7.5, 100 mM NaCl, 1 mM EDTA, 1% SDS, 2 mM Na₃VO₄), and subjected to SDS-PAGE, followed by transfer to Immobilon-FL PVDF membranes (Millipore). Membranes were blocked in 1% BSA/TBS containing 0.1% Tween20 for 30 min, and treated with primary antibodies in blocking buffer for 1 h, followed by treatment with IRDye-conjugated secondary antibodies (LI-COR). Images were obtained by using an ODYSSEY CLx quantitative IR fluorescent detection system (LI-COR), and quantified with Image Studio software Ver. 5.2 (LI-COR).