Fig. 3: The biological properties of UBOX5.

a, b UBOX5 and BIP are both induced by ER stress. a NIH3T3 cells were treated with the indicated ER stress inducers tunicamycin (Tu) and thapsigargin (Tg). (Upper) Endogenous UBOX5 mRNA abundance was quantified by qPCR in biological triplicates, normalised against mouse beta-actin transcript. Relative fold change of transcript is reported against control DMSO treatment. Error bars represent standard deviation. (b, Top) Protein abundance of endogenous mouse  UBOX5 in ER-stressed NIH3T3 cells. Beta-actin was used as loading control. Of note, UBOX5 mRNA and protein is induced in response to ER stress. (b, Bottom): Densitometric quantitation of UBOX5 bands. UBOX5 band intensity of each sample is normalised to corresponding beta-actin intensity. The amount of TG or TU used is indicated on the x-axis. P-values were generated from two-sided Welch’s t-test. Error bars represent standard deviation. Source data are provided as a Source data file. c Cellular Localization of UBOX5: Immunoblots of HEK293 cells transiently transfected with UBOX5 expression plasmid and treated with 0.7 uM Thapsigargin for 16 h. Cellular fractions are indicated above. Whole cell lysates were fractionated into cytoplasmic, endoplastic reticulum (ER) fractions and nuclear fractions by stepwise centrifugation. Indicated antibodies are shown. Positions of molecular weight markers are indicated on the left with arrows. GFP is used as transfection control, Calnexin is used as fractionation control for ER and nuclear fraction; Histone H2B is used as fractionation control for nuclear fraction. GAPDH is used as fractionation control for cytoplasmic and nuclear fractions. The experiment was repeated independently 3 times. Source data are provided as a Source data file. d, e The ability of UBOX5 to ubiquitinate BIP is not dependent on cellular stress. d MYC-tagged BIP, empty vector, UBOX5, or HA-tagged ubiquitin was co-transfected into HEK293 cells in the indicated combinations. 24 hours later, cells were treated with 0.7 μM Thapsigargin (TG) or DMSO for 16 h. TG is a known inducer of endoplasmic reticulum stress. 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. e 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. GAPDH was used as loading control.