TRIM37 interacts with and ubiquitinates centrobin. (A) Immunoblotting of centrobin in the indicated cell lines. Graph on right plots centrobin levels relative to the mean control value (n = 3). (B) Schematic of coexpression-based analysis of regulation of centrobin by TRIM37 shown in C–G. (C) Immunoblotting of crude extracts with the indicated antibodies. (D) Supernatant (S) and pellet (P) fractions immunoblotted with the indicated antibodies. α-tubulin fractionates into the supernatant, as expected for extracts prepared after cold treatment. Even under the denaturing conditions used, centrobin and Lig^mut TRIM37 were not consistently quantitatively recovered from the pellet. (E) Band signal intensity values from two independent experiments of total centrobin in crude extract (left), relative centrobin enrichment in pellet (middle), and relative Lig^mut TRIM37 enrichment in pellet; the Lig^mut TRIM37 pellet enrichment plot has a logarithmic y axis. Sup, supernatant. (F) Anti-Myc and Anti-FLAG immunoblotting of anti-Myc immunoprecipitates (IP). (G) Centrobin ubiquitination by TRIM37; a plasmid encoding HA-ubiquitin was included in the coexpression. The input shown is the crude extract. (H and I) FRAP analysis of Lig^mut TRIM37-mNG localized to condensates in TRIM37Δ cells. In H, multiple time point images and quantification of signal intensity along a 10-pixel-wide line is shown. The intensity value at the left most position before the condensate was used as the background and subtracted; after background subtraction, all pixel values were normalized by dividing by the average prebleach signal. In I, selected images are shown above the linescan plots. Images of the condensates from individual time-lapse movie frames were cropped and rotated before montaging. Scale bars, 1 µm. The experiment was performed five times with similar results; three examples are shown. α-Tubulin serves as a loading control in A, C, D, and G. high exp., higher exposure; low exp., lower exposure.