Pa1 10033

Cells grown on acid‐etched glass coverslips were washed with PBS and fixed with 3.7% formaldehyde in PBS buffer for 10 min. Cells were kept in blocking buffer (3% BSA in PBS) for 1 h and incubated for 2 h or overnight with primary antibodies diluted in 3% BSA/PBS buffer followed by 1‐h incubation with secondary antibodies (Table 2). Primary antibodies were against, α‐tubulin mouse (1:1,000; Sigma), α‐tubulin rabbit (1:800; 18251; Abcam), GFP (1:1,000; 6556; Abcam), GFP (1:1,000; sc‐9996; Santa Cruz Biotechnology), ALK rabbit (D5F3) (1:100; CST), ALK (31F12) mouse (1:100; CST), GRB2 rabbit (1:1,000; PA1‐10033; Invitrogen), GRB2 (1:100) mouse (C7; Santa Cruz Biotechnology), SOS1 mouse (1:100; MCA2887; Bio‐Rad;), PLCγ2 (Y759) rabbit (1:100; AP0785; ABclonal), PI3K p85β mouse (1:100; 1B180967; Abcam), c‐KIT (Y721) (1:100; 44‐494G; Thermo Fisher Scientific). Secondary antibodies were Alexa Fluor‐488 and ‐594 goat anti‐rabbit and goat anti‐mouse IgGs (1:200; Invitrogen). Imaging was performed on a Zeiss LSM880 + Airyscan Inverted confocal microscope using a 40× oil objective (numerical aperture, 1.4). Z‐stacks comprising of 10–20 ×0.3 µm sections were acquired. Images were analysed using ImageJ (v.2.0.0).

For dose response experiments, cells were serum‐starved for 24 h prior to harvesting and were then lysed using M‐PER mammalian protein extraction reagent (Thermo Fisher Scientific) containing a Halt™ protease inhibitor cocktail (Thermo Fisher Scientific) and Halt™ phosphatase Inhibitor cocktail (Thermo Fisher Scientific). Immunoblot analyses were carried out with anti‐phospho‐ALK (Y1604) (1:1,000, 3341; CST), anti‐ALK (1:1,000, D5F3; CST), anti‐ERK (1:1,000, L34F12; CST), anti‐phospho‐ERK (T202/Y204) (1:1,000, D13.14.E3; CST), anti‐AKT (1:1,000, 40D4; CST), anti‐phospho‐AKT (S473) (1:1,000, D9E; CST), anti‐STAT3 (1:1,000, 124H6; CST), anti‐phospho‐STAT3 (Y705) (1:1,000, 3E2; CST), GRB2 rabbit (1:1,000; PA1‐10033; Invitrogen), SOS1 mouse (1:1,000; MCA2887; Bio‐Rad;), PLCγ2 rabbit (Y759) rabbit (1:1,000; AP0785; ABclonal), PI3K p85β (1:1,000; 1B180967; Abcam), c‐KIT (Y721) (1:1,000; 44‐494G; Thermo Fisher Scientific), anti‐calnexin (1:1,000, C5C9; CST), anti‐DCP1B (1:1,000, D2P9W; CST), GAPDH (1:2,000, ab37168; Abcam) and anti‐α‐tubulin (1:2,000, T5168; Sigma) antibodies. Secondary antibodies were rabbit or mouse horseradish peroxidase‐conjugated secondary antibodies (1:1,000; Amersham). The blots were visualised using the Pierce™ ECL Western blotting substrate (32106; Pierce).