Fig 2: Immunoblotting validation of key proteins centering on cell membrane structure. A Automated capillary Western blot validation of ANXA1, GRB2, and ANXA4. B The expression of ANXA1 (t = 14.216, df = 4, P < 0.001), GRB2 (t = 10.805, df = 4, P < 0.001), and ANXA4 (t = 10.118, df = 4, P = 0.009) was quantified according to the corresponding bands. GAPDH was used as the internal reference marker and loading control for normalization. These data were presented as mean ± SD of three independent experiments. NS > 0.05, **P < 0.01 and ***P < 0.001, compared with the corresponding control group

Fig 3: SCIMP-TLR interaction in macrophages. Primary BMMs were treated with the TLR2 ligand Pam3CSK4 (A) or the TLR4 ligand LPS (B), cell extracts were then used for immunoprecipitation with a SCIMP antibody, and immunoblotting detected endogenous TLR2 or TLR4, Lyn and a phospho-tyrosine antibody detected phospho-SCIMP. All data are representative of three experiments. C, GST-SCIMP effector-based pull-downs were performed in LPS-treated BMMs using site-specific phosphotyrosine probes (GST-Grb2-SH2, GST-Csk-SH2, and GST-SLP65-SH2) to detect SCIMP phosphorylated at sites at Y58, Y96, and Y120, respectively, shown on three separate gels. BMM lysates represent the input samples used for these pull downs immunoblotted to detect proteins of interest. BMMs, bone marrow–derived macrophages; LPS, lipopolysaccharides; TLR, Toll-like receptor.

Fig 4: RET::GRB2 fusion is detected as a somatic event in a pheochromocytoma(A) Representation of the region spanning the RET exon 18 (left panel) and GRB2 exon 3 (right panel) in a pheochromocytoma predicted to carry the RET::GRB2 fusion, indicating the number of spanning reads (n = 18) and junction reads (n = 492) detected by RNA-seq. Chromosome (chr) location is indicated for each gene.(B) Chromosomes (chrs) plotted as ideograms around the outside of the circle (CIRCOS) plot depicting the putative recombination between RET (chr10) and GRB2 (chr 17). The thickness of the connecting line represents the depth of read coverage.(C) Hematoxylin and eosin (H&E) staining of the primary composite tumor comprised of a predominant pattern of pheochromocytoma (right) combined with a focal area of ganglioneuroma (left); magnification 100×, scale bar, 100 µm (inset, 400×, scale bar, 20 µm).(D) Agarose gel of PCR products spanning the RET::GRB2 fusion transcript in tumor but not matched blood (leukocyte) cDNA; two other paired blood/tumor cDNA samples from patients with a RET mutation and a SDHB germline mutation, respectively, show no product (top); housekeeping gene TBP is shown (bottom). These results were repeated three times.(E) Sanger sequencing of the product shown in (D), displaying RET sequence (exon 18) merged with GRB2 (exon 3). This experiment was repeated three times.(F) RET (left) and GRB2 (right) mRNA expression obtained from high-throughput sequencing (HT-seq) counts of RNA-seq from 30 pheochromocytomas/paragangliomas (PPGLs; dots), depicting an exon within the fusion (x axis) and one outside the fusion (y axis). RET::GRB2-positive tumor is indicated in blue.(G) DNA-based quantitative real-time PCR assay for copy-number assessment of the RET and GRB2 genes using primers targeting the area within (x axis) or outside the fusion (y axis) of a set of 14 PPGLs of various genotypes (dots). This experiment was repeated three times in duplicate samples.

Fig 5: Validation of the RET::GRB2 fusion protein in the pheochromocytoma and in vitro(A) Diagram of wild-type (WT) RET, displaying relevant domains as indicated (CadLD, cadherin-like domains; CRD, cysteine-rich domain; TM, transmembrane domain; TKD, tyrosine kinase domain). The two main RET isoforms diverge at amino acid (aa) 1,063 (*) with either 51 (RET51) or 9 (RET9) distinctive aa at the C terminus; WT GRB2 contains two SH3 domains flanking one SH2 domain. The RET (R1013) and GRB2 (V27) breakpoint sites are indicated; full-length RET::GRB2 fusion spans 1,204 aa.(B) Western blot of protein lysates from PPGLs carrying mutations in RET, SDHB, VHL, and EPAS1 genes, and the RET::GRB2 fusion, probed with phosphorylated (P) RET (Y905) and two distinct total (T) RET antibodies directed at the extracellular, N-terminal (N-term) region around D320 and C-terminal (C-term) region beyond aa 1,100. GRB2 shows both the WT and fusion product, P- and T- AKT; ERK1/2, ß-actin is a loading control; two technical replicates were performed.(C) SH-SY5Y cells stably expressing RET9, RET51, RET::GRB2, and a control vector, probed with RET, GRB2, and a-tubulin as a loading control; three biological replicates were performed.(D) SH-SY5Y cells expressing RET::GRB2 or RET51 were starved of serum for 3 h and exposed to 100 ng/mL GDNF (+) or vehicle (-) for 10 min, and lysates were probed with P-RET Y905 or T-RET; a-tubulin is a loading control; experiments were repeated three times.(E) SH-SY5Y cells expressing RET::GRB2, RET 9, or RET51 constructs were treated with GDNF as in (D). Lysates were probed with GRB2 and T- and P- AKT, ERK1/2, T RET, and RET phosphorylated at Y1062, a region excluded from the RET::GRB2 fusion; a-tubulin is a loading control; experiments were repeated three times.(F) Confocal microscopy of HEK293T cells expressing WT, mutant (C634R) RET, or RET::GRB2 fusion, labeled with a tag antibody in green (MYC for WT and C634R RET or hemagglutinin [HA] for RET::GRB2 fusion) and a membrane marker, Na/K ATPase (red). Nuclei are stained with DAPI (blue). A merged image is shown in the right panels. Scale bar: 10 µm.(G) Quantification of the colocalized signals between RET and the Na/K ATPase using ImageJ from multiple independent images (n = 10–13 cells/genotype, two biological replicates). One way ANOVA, p = 0.048; *p = 0.02, two-tailed t test; **p = 0.0042, two-tailed t test.(H) Uniform manifold approximation and projection (UMAP) plot of RNA-seq data from pheochromocytomas/PPGLs of our cohort (n = 30, UTHSA) and TCGA (n = 178), color-coded by genotype; gray symbols are tumors with unknown mutations; RET::GRB2 fusion (red triangle).