Phospho akt serine 473 xp

The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); MITF (1:1000; #12590), Her3/ERBB3 XP (1:1000; #12708), Phospho-Her3/ERBB3-Tyrosine-1289 (1:1000; #4791), FOXD3 (1:1000; #2019), SOX10 (1:1000; #14374), Akt (1:2000; #9272), Phospho-Akt-Serine-473 XP (1:2000; #4060), and Histone H3 (1:3000; #4499) was used as loading control. Secondary antibody against rabbit (1:5000; P0448) was purchased from Dako (Agilent Technologies, Glostrup, Denmark). The small molecular inhibitor vemurafenib (Plexxikon 4032) was obtained from Selleck Chemicals (Houston, TX, USA). The NRG1-beta ligand was purchased from ImmunoTools GmbH (Friesoythe, Germany). The NRG1-beta ligand was used to enhance the pERBB3 (T1289) signal, as our Phospho-Her3/ERBB3-Tyrosine-1289 antibody did not detect pERBB3 (T1289) levels without NRG1-beta addition (see also Supplementary Figure S3A-S3B). The reason for choosing 15min of incubation time and a NRG1-beta ligand concentration of 10ng/ml was due to a previous study where they showed efficient activation of the ERBB3 receptor in the WM115 cell line under these conditions [18 (link)].

Antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); MITF (1:1000; #12590), Her3/ERBB3 XP (1:1000; #12708), AXL (1:1000 #8661), SOX10 (1:1000; #14374), Phospho‐Akt‐Serine‐473 XP® (1:2000; #4060), Phospho‐p44/42 (ERK1/2) XP® (1:2000; #4370S) and Histone H3 (1:3000; #4499) was used as a loading control. Secondary antibody against rabbit (1:5000; P0448) was purchased from Dako (Agilent Technologies, Glostrup, Denmark). The small molecular inhibitors vemurafenib (Plexxikon 4032), AZD8931 (Sapatinib) and R428 (BGB324/bemcentinib) were obtained from Selleck Chemicals (Houston, TX, USA). Recombinant human NRG1‐beta protein‐ligand was purchased from ImmunoTools GmbH (Friesoythe, Germany). Recombinant human GAS6 protein‐ligand was purchased from R&D Systems (Minneapolis, USA).

Western Blot analysis was performed as previously described [40 (link)]. The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); MITF (1:1000; #12590), SOX10 (1:1000; #14374, Phospho-Akt-Serine-473 XP (1:2000; #4060), Phospho-ERK–Thr202/Tyr204) XP (1:3000; #4370), PTEN XP (1:1000; #9188) and Histone H3 (1:3000; #4499) was used as loading control. Anti-HA-tag (12CA5) was purchased from Roche. Secondary antibody against rabbit (1:5000; P0448) was purchased from Dako (Agilent Technologies, Glostrup, Denmark). Uncropped, unedited blots from Figure 1A, Figure 4A,C,E,F,I as well as for Figure S1I and Figure S2A can be found in Figure S5.