Fig 2: UBQLN1, UBQLN2, and UBQLN4 exhibit different phase behaviors. (A) Domain architectures of UBQLN1, UBQLN2, and UBQLN4. The proline-rich repeat (Pxx) region is unique to UBQLN2. (B) Brightfield microscopy shows 200 μM solutions of the three proteins in buffer containing 20 mM sodium phosphate, 200 mM NaCl, 0.5 mM TCEP, and EDTA (pH 6.8) after incubation at 30°C for 5 min. Imaging was done at the coverslip. Scale bar, 10 μm. (C) Phase diagrams determined from csat measurements as a function of temperature. Phase separation is observed above these coexistence lines. Error bars represent SD over at least four trials from two different proteins preps.

Fig 3: shUbqln1 mES cells Differentiation into Neuronal or Epithelial-like cells. Schematic presentation of UBQLN1 and UBQLN2 having different cell type specific functions. (A) Schematic of doxycycline inducible UBQLN1 shRNA differentiation into mouse embryonic stem cell neuronal (mESN) and mouse embryonic stem cell epithelial (mESE) cells. (B) Representative microscopy images of unmanipulated mES cells and mES cells following doxycycline induction. (C) Representative t-Distributed Stochastic Neighbor Embedding (t-SNE) plots of unmanipulated neuronal/differentiated neuronal and unmanipulated epithelial/differentiated epithelial cells. Neuronal cells revealed identical transcriptional profile throughout the cell populations (green vs black spots) while loss of UBQLN1 found to alter differentiation of epithelial cells (blue vs red spots). (D) Schematic presentation of UBQLN1 and UBQLN2 having different cell type specific functions. UBQLNs has N-terminal UBL domain (ubiquitin-like), C-terminal UBA domain (ubiquitin-associated) and STI regions in the middle. Ubiquilin2 has an additional collagen-like domain between the fourth STI and UBA domains.

Fig 4: RNAseq comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in A549 and SK-N-BE(2) cells. (A) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in A549 cells following RNAseq analysis. (B) Representative list of top 10 genes down-regulated specifically by siUBQLN1. (C) Representative list of top 10 genes down-regulated specifically by siUBQLN2. (D) Representative list of top 10 genes up-regulated specifically by siUBQLN1. (E) Representative list of top 10 genes up-regulated specifically by siUBQLN2. (F) Representative bar graph showing confirmation of genes that were significantly altered following loss of UBQLN1 in A549 and HOP62 cells. (G) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in SK-N-BE(2) cells following RNAseq analysis. (H) Representative Venn diagram showing comparisons of overlapping genes differentially expressed upon UBQLN1 and UBQLN2 knockdown in A549 and SK-N-BE(2) cells following RNAseq analysis.

Fig 5: The axon guidance gene beat-1b is a UBQLN24XALS suppressor.(A) Schematic of the beat-1b gene locus. (B) Representative eye phenotypes of GMR>UBQLN24XALS flies harboring the indicated deficiency (Df) alleles. (C) UBQLN2 expression levels and RIPA solubility in whole heads of GMR>UBQLN24XALS/Exel6038 and GMR>UBQLN24XALS/CyO flies. (D) Representative eye phenotypes of GMR>UBQLN24XALS flies expressing the indicated shRNAs. Eye degeneration scores are displayed below each eye image (B, D). (E, F) Knockdown of Beat-1b or Beat-1c does not inhibit UBQLN2 expression in GMR>UBQLN24XALS flies. (F) Quantification of UBQLN2 expression normalized to β-tubulin. The bars represent mean with SEM of triplicate samples. Unpaired t-test was used for statistical analysis. . Figure 5—source data 1.Uncropped Western blot images corresponding to Figure 5C, E, F.