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Recombinant analogue of human SLURP-1 (1 mg/ml) was coupled to NHS-activated Sepharose 4 Fast Flow resin (Cat# 17-0906-01, GE Healthcare) according to the manufacturer’s manual. The empty resin blocked by 500 mM ethanolamine was used as a negative control. The membrane fraction of A549 cells (5 × 10⁷ cells per sample) was solubilized in 2% Triton X-100 (Cat# A4975, Panreac), diluted 10 times with TBS buffer [100 mM Tris (141940.0914, Panreac), 150 mM NaCl (141659, Panreac), pH 8.0], and incubated with the resin for 1 h in TBS. After that, unspecific bound proteins were sequentially washed out from the resin with five volumes of TBS, five volumes of TBS + 1 M NaCl + 0.5% Triton X-100, and five volumes TBS + 0.5% Triton X-100. The specifically bound proteins were eluted by five volumes of 200 mM Glycine (131340, Panreac) (pH 2.6) into non-reducing PAGE loading buffer for detection of EGFR and in reducing PAGE buffer for detection of α7-nAChR and PDGFRα. Western blotting was used to detect α7-nAChR (primary Abs ABIN5611363, Antibodies Online, 1:1000 and secondary Abs 111-035-003, Jackson Immunoresearch, 1:5000), EGFR (primary Abs sc-120, Santa Cruz, 1:1000 and secondary Abs 715-035-150, Jackson Immunoresearch, 1:5000) and PDGFRα (primary Abs ABIN5611263, Antibodies Online, 1:1000 and secondary Abs 715-035-150, Jackson Immunoresearch, 1:5000).

For cross-linking^{60 (link)}, Protein A-Sepharose 4B beads (P9424, Merck Millipore) were re-suspended in a column in 50 mM Tris pH 7 to make a 50% slurry, and incubated with EGFR antibody 528 (sc-120, Santa Cruz Biotechnology) or normal mouse IgG (sc-2025, Santa Cruz Biotechnology) overnight at 4 °C. The beads were then washed 3× with 0.2 M sodium borate pH 9, and incubated with a freshly made solution of 0.2 mM dimethyl pimelimidate in sodium borate for 40 min at RT. The beads were washed once with 0.2 M ethanolamine pH 8 (quenching solution), and further incubated for 2 h with this solution. Uncoupled antibodies were removed with 3X wash in 0.58% acetic acid in 150 mM NaCl. The beads were then transported into Eppendorf tubes.

A total of 2 × 10⁵ cells were plated on 6 well-plates and cultured in RPMI containing 1% FBS. After 48h, cells were isolated by trypsinization using trypsin-versene EDTA solution, then incubated with an anti-EGFR antibody (EGFR-AF488, 1/100, SC-120, Santa Cruz) for 30 min at 4°C before analysis on flow cytometer (Calibur II or ARIA II, Becton Dickinson).

The ability of binase to interact with EGFR and RAS proteins was assessed by Abcam's Immunoprecipitation Kit (ab206996; Abcam, USA). BT-20 cells and KRAS-transformed ROSE 199 A2/5 cells were seeded by 150000 cells/well in a 6-well plate and grown for 24 h (90% confluence). Afterwards, cells were lysed with nondenaturing lysis buffer (Abcam, USA) containing a protease inhibitor cocktail (Invitrogen, USA). Total protein content was quantified with Pierce Coomassie (Bradford) Protein Assay Kit (Thermo Scientific, USA).
For immunoprecipitation, 20 μg of binase protein was incubated with 1000 μg of each cell protein lysate for 15 min at 37°C on a shaker with 200 rpm and kept on ice. Then, 1 μg primary mouse monoclonal antibodies against EGFR (sc-120; Santa Cruz, USA) or 1 μg primary mouse polyclonal anti-IgG antibodies as a control (sc-2025; Santa Cruz, USA) were added to BT-20 cell protein lysates and incubated at 4°C overnight. In the case of ROSE 199 A2/5, 1 μg primary mouse monoclonal antibodies against pan Ras (sc-166691; Santa Cruz, USA) instead of anti-EGFR antibodies were added. The immunocomplexes were precipitated with 25 μL prewashed Protein A/G Sepharose beads for 1 h at 4°C. Beads were collected and loaded on 7% or 16% SDS-PAGE for western blot analysis as described under 4.4.

Total proteins were extracted in RIPA lysis buffer with protease inhibitors and incubated on ice for 30 min. The cell lysate supernatant was supplemented with 4 × loading buffer and boiled for 10 min. Proteins were separated by 8% or 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes (0.45 µm, Bio-Rad), and nonspecific binding was blocked with 1% bovine serum albumin (BSA) for 1 h at room temperature. The membranes were incubated with specific primary antibodies overnight at 4 °C and with secondary antibodies for 1 h at room temperature. The primary antibodies used in this study include Kremen2 (SAB1305119, Sigma), STAT3 (ab119352, Abcam), p-STAT3 (Y705) (ab76315, Abcam), JAK2 (ab108596, Abcam), p-JAK2 (Y1007 + Y1008) (ab32101, Abcam), LRP6 (#2560, CST), p-LRP6 (Ser1490)(#2568, CST), β-catenin (ab32572, Abcam), EGFR (sc-120, Santa Cruz), p-EGFR (Tyr1068) (#2234, CST), SOCS3 (ab16030 and ab236519, Abcam), c-Myc (ab32072, Abcam), CyclinD1 (ab226977, Abcam), PI3K (#4257, CST), p-PI3K (Y607) (ab182651, Abcam), AKT (#9272, CST), p-AKT (Ser473) (#4060,CST), GAPDH (60,004–1-Ig, Proteintech), Flag (F1804, Sigma), HA (51,064–2-AP, Proteintech).