Near infrared fluorescence

Cells for protein analysis were lysed in RIPA buffer (20 mM Tris-HCl, 37 mM NaCl₂, 2 mM EDTA, 1% Triton X-, 10% glycerol, 0.1% SDS, and 0.5% sodium deoxycholate) with phosphatase and protease inhibitors. For immunoprecipitations, cells were lysed in IP lysis buffer (100 mM NaCl, 25 mM Tris, 1% Triton X-, 10% glycerol, with phosphatase and protease inhibitors) and immunoprecipitated with 25 μL of 50% slurry of protein Recombinant protein G-Sepharose 4B (Thermo Fisher). Immunoprecipitates were washed three times with lysis buffer, once with high salt (500 mM NaCl), and once more with lysis buffer. Protein concentration in lysates was determined by using Protein Assay Kit (Bio-Rad). Cell extracts and immunoprecipitated proteins were denatured, subjected to SDS-PAGE, transferred to PVDF membranes (GE Healthcare). After blocking with 5% nonfat dry milk in Tris-buffered saline and 0.1% Tween (TBS-T), the membranes were incubated with the specific antibodies (as listed in key resources table) overnight at 4°C. After 2 h incubation with the appropriate horseradish peroxidase-conjugated antibodies, the immune complexes were detected by chemiluminescence (Thermo Fisher) or Near-infrared fluorescence (LI-COR).

Cells were lysed in RIPA buffer (10mM Tris-HCl [pH 8.0], 150mM NaCl, 1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100) with protease and phosphatase inhibitors (Roche). Protein concentrations were measured using the DC Protein Assay Kit (Bio-Rad). SDS–PAGE and protein transfer were performed using Mini Gel Tank and Mini Blot Module (Life Technologies). Immunoblotting was detected using near-infrared fluorescence (LI-COR) and the Odyssey CLx imager (LI-COR). Quantitative analysis of immunoblots was performed using Image Studio Lite software (LI-COR). The following primary antibodies were used: EREG (1:1000) [Cell Signaling Technologies (CST), 12048], HB-EGF (1:10000) (Abcam, ab185555), p-EGFR (Y1068) (1:1000) (CST, 3777), EGFR (1:1000) (CST, 4267), p-PAK1 (S199/204)/p-PAK2 (S192/197) (1:500) (CST, 2605), PAK1 (1:300) (Santa Cruz, sc-882), p-Erk1/2 (T202/Y204) (1:2000) (CST, 4370), Erk1/2 (1:1000) (CST, 4695), p-Akt (S473) (1:1500) (CST, 4060), pan-Akt (1:1000) (CST, 4691), α-tubulin (1:10000) (Sigma, T6074), and β-actin (1:20000) (Sigma, A1978). To analyze phosphorylation of proteins, membranes were probed with phospho-specific antibodies first, stripped with NewBlot IR Stripping Buffer (LI-COR) and then reprobed with pan antibodies.