Fig. 2: UBOX5 has E3 ubiquitin ligase activity.

a A UBOX5 chimeric construct (UBOX5-UBD) where the UBOX5 open reading frame was fused by a flexible linker to a FLAG-tagged ubiquitin binding domain was generated. FLAG-tagged UBOX5 (3 lanes on the right) or FLAG-tagged UBOX5-UBD construct (4 lanes on the left) and HA-tagged ubiquitin was co-transfected into HEK293 cells. After the first immunoprecipitation of lysates by anti-FLAG antibody, 20% of eluates was kept for analysis. Eluate from the UBOX5-UBD was further immunoprecipitated with anti-HA antibody to enrich for ubiquitinated proteins. To serve as antibody specificity control (negative controls), lysates were mock immunoprecipitated with mouse immunoglobulin. Immunoblotting of BIP was performed on inputs and eluates as indicated. Bands corresponding to ubiquitinated BIP and ubiquitinated UBOX5-UBD chimeric protein are indicated by vertical lines, while the unmodified proteins are indicated by arrows on the right. Positions of the molecular weight markers are indicated by arrows on the left. IB: FLAG and IB:HA are shown in Supplementary Fig. 13. The experiment was repeated independently 3 times. Source data are provided as a Source data file. b MYC-tagged BIP, empty vector, UBOX5, or HA-tagged ubiquitin was co-transfected into HEK293 cells in the indicated combinations. 24 h later, cells were treated with 0.7 uM Tharpsigargin for 16 h, and then further treated with MG132 (a proteasomal inhibitor) for 6 h as indicated. Cells were then harvested and a MYC immunoprecipitation was performed on the input lysates. Eluates were immunoblotted with antibodies against HA to assess the extent of BIP ubiquitination. The membrane was then stripped and a MYC immunoblot was performed to assess immunoprecipitation efficiency. Ubiquitination of BIP was only observed when UBOX5 was expressed. The degree of ubiquitination of BIP did not appear to differ with the addition of MG132, a proteasomal inhibitor, suggesting that the ubiquitinated BIP was not degraded by the proteasome pathway. The experiment was repeated independently 3 times. Source data are provided as a Source data file. (c) UBOX5 or its empty vector contains a GFP open reading frame, which allows for assessment of transfection efficiency by assessing GFP abundance in input lysates. Human UBOX5 immunoblots were used to verify expression of UBOX5. GAPDH was used as loading control. The experiment was repeated independently 3 times. Source data are provided as a Source data file.