Extended Data Fig. 9: Neomycin depletes anti-PIP2 and anti-PIP3 immunostaining.

a, Neomycin was used to test the specificity of anti-PIP2 immunofluorescence. Neomycin binds to the head groups of phosphorylated phosphoinositides. Cells were co-immunostained for PIP2 (green) and SON. Either neomycin (2 mg/ml) or vehicle (PBS) was added to both the pre-antibody incubation with 2% FBS and during the primary antibody incubation. PIP2 images are displayed with increased brightness for cytoplasmic granules since there is less PIP2 in the cytoplasm than the nucleus. A representative image is displayed for each immunostain. Scale bar, 5 μm (n = 2). b, Metaplot of PIP2 signal proximal to condensates imaged in a. PIP2 signal was measured in the area surrounding each condensate’s center. The median protein (red) and PIP2 (green) signal intensity across all condensate images is plotted. Each metaplot represents one representative experiment. Both the vehicle and neomycin treatments for each experiment were performed at the same time using the same imaging and display conditions. Neomycin treatment depletes the PIP2 signal observed in condensates (n = 2). c, To determine if anti-PIP3 immunofluorescence in condensates is specific to PIP3, neomycin was used to deplete anti-PIP3 immunofluorescence. Cells were co-immunostained for PIP3 (green) and G3BP1 as described in Fig. 6c. In the presence of neomycin, there is still significant amount of background signal across the cell, but the enrichment of PIP3 in stress granules and P bodies is no longer observed, suggesting that signal is specific. A representative image is displayed for each immunostain. Scale bar, 5 μm (n = 2). d, Metaplot of PIP3 signal proximal to condensates imaged in c. PIP3 signal was measured in the area surrounding each condensate’s center. The median protein (red) and PIP3 (green) signal (intensity) across all condensate images from an experiment is plotted for immunostains with or without neomycin (as indicated), with the distance from the center of each condensate indicated on the x-axis and y-axis. Both the vehicle and neomycin treatments for each experiment were performed at the same time using the same imaging and display conditions. PIP3 signal in condensates is depleted by neomycin (n = 2).