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. 2015 May 26;26(11):2789–2799. doi: 10.1681/ASN.2014080846

Figure 6.

Figure 6.

Inhibition of YB-1 ubiquitination by aPC depends on OTUB1. (A) and (B) HR (6 hours of hypoxia and 6 hours of reoxygenation; C: normoxic control) decreases OTUB1 levels in BUMPT cells in vitro. Treatment of BUMPT cells with aPC (20 nM) conserves OUTB1 expression despite HR. Representative IBs (A) and bar graph summarizing the results (B). (C) Ubiquitination in BUMPT cells stably expressing empty vector (C) or an expression construct for flag tagged OTUB1 (OTUB1+). Overexpression of OUTB1 reduces HR-induced protein ubiquitination (top; ubiquitin IB), maintains YB-1 expression (middle; β-actin as loading control), and reduces YB-1 K48–linked ubiquitination (bottom, αK48 IB: IP of YB-1 followed by K48-linked ubiquitin IB; YB-1 IB shown as input control). Flag IB reflecting efficient overexpression of flag-tagged OTUB1. (D) OTUB1 deficiency impairs aPC’s effect on ubiquitination and YB-1 expression. The aPC mediated reduction of protein ubiquitination (C, top) and YB-1 K48-linked ubiquitination (C, bottom) and the sustained YB-1 expression (C, middle, β-actin as loading control) are impaired in stable OTUB1 knockdown tubular cells (shRNA OTUB1) when compared with control transfected cells (shRNAc). YB-1 K48–linked ubiquitination is determined by IP of YB-1 followed by K48 immunoblotting (αK48 IB); YB-1 IB shown as input control. Mean±SD value of three independent experiments (B); *P<0.05, (ANOVA); representative IBs of at least three independent experiments [(A), (C), and (D)]. IB, immunoblot; IP, immunoprecipitation.