Fig 2: Proteomic analysis reveals differential enrichment of proteins in SGs and RLBs.(A) Volcano plot showing differentially enriched proteins following APEX proximity labeling in HEK293T cells expressing G3BP1-APEX2-GFP fusion protein. Cells were treated with sodium arsenite (SA, 1.0 mM for 1 h) or poly(I:C) (1.0 μg/mL for 4 h) to induce granule formation before labeling. The x-axis shows the log2 fold-change of the APEX abundances, normalized to input, for SA compared to poly(I:C). The y-axis indicates the −log10 p-value for the comparison of normalized APEX abundances for the three biological replicates of SA and poly(I:C) treatments. Proteins significantly enriched in SGs (left, green) or RLBs (right, magenta) are highlighted. Proteins validated by our proteomic analysis or identified in previously published datasets are labeled in the figure. (B) Venn diagram comparing proteins enriched in SGs and RLBs. Proteins that are unique to SGs (left, green), unique to RLBs (right, magenta), or shared between the granules (intersection) are indicated. Proteins further characterized in this study are highlighted by asterisks. (C-D) Validation of APEX-identified proteins was performed in A549 cells treated with sodium arsenite (SA, 0.5 mM for 1 h), poly(I:C) (0.5 μg/mL for 6 h) or infected with Ad5 WT, ΔVA or ΔE4 viruses at an MOI of 10 for 40 h. G3BP1 staining (green) was used as a marker for granule assembly. (C) Co-staining with FXR1 or UBAP2L (magenta) confirms recruitment of proteins to both SGs and RLBs. (D) Staining for eIF4G1 or FAM120A (magenta) shows proteins recruited to SGs, but absent from RLBs. Nuclei were stained with DAPI (blue). Scale bar = 15 μm (main panels), 2 μm (cropped panels).