Sc 9104

HeLa cell lysates, 20 μg/sample, were run on a 4–12% Bis-Tris polyacrylamide gels using MES as a trailing ion in the buffer (Invitrogen). Proteins were transferred to a nitrocellulose membrane (Whatman) using Towbin buffer with 20% (v/v) methanol in a semi-dry transfer apparatus (Hoefer) and reversibly stained with Ponceau S for loading control. Membranes were incubated in TBS buffer containing 0.01% Tween-20 and 5% (w/v) skim milk powder (Fluka), containing the primary antibodies. The following antibodies were used: ER stress antibody sampler kit (all 1:1000; #9956, Cell Signaling Technology; including secondary antibodies used here), anti-MAFF (1:500; AV38984, Sigma-Aldrich), anti-eIF4G1 (1:1000; 2858S, Cell Signaling Technology), anti-tubulin (1:1000; sc-9104, Santa Cruz Biotechnology). Puromycin incorporation was detected using the 12D5 mouse monoclonal antibody (MABE343, Millipore) at a 1:5000 final concentration. The quantification of gel bands was performed using the image processing program ImageJ (http://imagej.nih.gov/ij) (Schneider et al., 2012 (link)).

Cell lysates were analysed by western blot, using a rat anti-Blimp1 monoclonal antibody (Santa Cruz, sc-130917, 1:500) and rabbit anti-Tubulin polyclonal antibody (Santa Cruz, sc-9104, 1:1,000). All uncropped western blots can be found in the Supplementary Fig. 7.

Protein expression of NTRK2 was evaluated on 2 KIT^WT/PDGFRA^WT/SDH^WT/RAS-P^WT GIST and 8 KIT or PDGFRA or SDH mutant GISTs, of which fresh-frozen tissues were available. Tissue were disrupted in RIPA buffer (Sigma-Aldrich) supplemented with proteases inhibitors and lysed for 1 h with gentle agitation at 4°C. Lysates were centrifuged at 13,000 × g for 15 min at 4°C and supernatants were stored at −80°C. Protein concentrations were determined with the BCA protein assay (Pierce, Rockford, IL). Twenty micrograms of protein were resolved on a 8% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes. Nonspecific binding sites were blocked by incubation in blocking buffer (PBS containing 0.1% Tween-20 with 5% w/v milk) for 1 h at room temperature. Membranes were incubated overnight at 4°C, with the following primary antibodies: rabbit polyclonal TRKB antibody (ab18987 Abcam 1:500), and rabbit polyclonal β-Tubulin antibody (sc-9104 Santa Cruz Biotechnology, Santa Cruz, CA, 1:500). Then, membranes were washed and incubated with peroxidase conjugate secondary antibodies for 1 h at room temperature. Antigens were revealed using Enhanced Chemiluminescence Reaction (ECL Select, Amersham Pharmacia Biotech, Les Ulis, France).

Remodelin (S7641), α-MSH (M4135), arbutin (A4256), kojic acid (K3125), and l-DOPA (333786) were purchased from Selleckchem (Houston, TX, USA) and Sigma-Aldrich (St. Louis, MO, USA). Antibody against MITF (#12590) was obtained from Cell Signaling Technology (Danvers, MA, USA). Antibodies against tyrosinase (sc-7833) and β-tubulin (sc-9104) were supplied by Santa Cruz Biotechnology (Dallas, TX, USA).

The following antibodies were used: anti‐MCU (1:1,000, HPA016480, Sigma‐Aldrich), anti‐β‐tubulin (1:5,000, sc9104, Santa Cruz), anti‐HIF‐1α (1:500, 610958, Becton Dickinson), and anti‐hydroxy‐HIF‐1α (1:1,000, 3434, Cell Signaling).