Ab32562

Cells were lysed in 1× RIPA buffer (Cell Signaling Technology) with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). The lysate was incubated on ice for 30 min and cleared by centrifugation at 13,000 Rcf for 30 min at 4 °C. Immunoprecipitation was performed with anti-GD2 mAb, anti-GD3 mAb, or normal mouse IgG (sc-2025; Santa Cruz) in the presence of protein A Sepharose (Dynabeads^® Protein A, #10002D, Invitrogen) 4 h (or overnight) at 4°C in a rocking incubator. Resulting immune-complexes were subjected to immunoblotting. Blots were probed with specific primary Abs, then incubated with appropriate HRP-conjugated secondary Ab for 1 h. Bands were visualized with ECL reagents (PerkinElmer).
Primary Abs used for immunofluorescence staining were EGFR (ab32562; Abcam), c-Met (sc-10), integrin β1 (sc-9936) (Santa Cruz), anti-fibronectin (rabbit, F1, ab32419, Abcam), anti-vimentin (rabbit, V9, sc-6260, Santa Cruz), anti-N-cadherin (mouse, 32/N-Cadherin, #610921, BD Bioscience), anti-phospho EGFR Tyr1173 (rabbit, 53A5, #4407, Cell signaling), anti-ERK1/2 (rabbit polyclonal, sc-94, Santa Cruz), anti-phospho ERK1/2 Tyr204 (mouse, E4, sc-7383, Santa Cruz), anti-Akt (mouse, 40D4, #2920, Cell signaling), anti-phospho Akt Ser473 (rabbit, D9E, #4060, Cell signaling) and anti-GAPDH (rabbit polyclonal, G9545, Sigma-Aldrich).

Cells were plated in 12‐well Corning^® (Corning, NY, USA) plates using coverslips (12 mm Ø) and fixed for 15 min in 4% paraformaldehyde when highly confluent. Then, cells were permeabilized in PBS 1x‐0.1% Triton and blocked using PBS 1x‐10% Goat serum for 30 min. Primary antibodies SOX9 (1 : 100, ab76997; Abcam, Cambridge, UK), smooth muscle actin (SMA, 1 : 100, RB‐9010‐R7; ThermoFisher Scientific, Waltham, MA, USA), EGFR (1 : 50, ab32562; Abcam), p75 (1 : 100, AB‐N07; ATSbio, Carlsbad, CA), and S100B (1 : 1000, Z031129; Dako, Glostrup, Denmark) were diluted in PBS‐1% Goat serum and incubated overnight at 4 °C. Secondary antibodies Alexa Fluor 488 goat anti‐mouse (1 : 1000, A11029; Invitrogen, Waltham, MA, USA), Alexa Fluor 488 donkey anti‐rabbit (1 : 1000, 711‐545‐152; Jackson ImmunoResearch, Philadelphia, PA, USA), and Alexa Fluor 568 goat anti‐rabbit (1 : 1000, A11036; Invitrogen) were diluted in PBS‐10% Goat serum and incubated for 1 h at RT. After washing three times with PBS 1x, DAPI diluted in PBS (1 : 1000, 62248; ThermoFisher Scientific) was added for 10 min and then washed three times, and finally, coverslips were mounted in Immu‐Mount (9990402; ThermoFisher Scientific). Images were acquired using a Nikon Eclipse 80i fluorescence microscope with nis‐Elements Microscope Imaging Software and analyzed using imagej fiji software (Lexington, KY, USA).

Cells grown on glass coverslips were fixed in 4% paraformaldehyde in PBS for 15 min at room temperature (RT), and permeabilized with 0.5% Triton X-100 in PBS for 5 min. Fixed cells were blocked with 5% BSA in PBS for 30 min, incubated overnight at 4°C with primary Ab, washed, incubated 1 h at RT with secondary Ab, and counterstained with DAPI (Pharmingen). Coverslips were mounted with glycerol mounting medium (Dako) and sealed with clear nail polish. Fluorescence images were obtained by confocal immunofluorescence microscopy (model TCS SP8; Leica).
Primary Abs used for immunofluorescence staining were anti-GD2 (mouse, Clone 14.G2a, #554272, BD Biosciences), anti-GD3 (mouse, clone R24, ab 11779, Abcam), anti-EGFR (rabbit, ab32562; Abcam), and anti-c-Met (rabbit, sc-10, Santa Cruz). Secondary Abs used were Alexa555-conjugated donkey anti-mouse IgG and donkey anti-rabbit IgG (Jackson ImmunoResearch) and Alexa488-conjugated donkey anti-rabbit IgG and donkey anti-mouse IgG (Invitrogen).