Figure 6.

PIPSL proteins lack PIP5 kinase activity, and bind cellular ubiquitinated proteins. (A) C-terminally HA-tagged human PIP5K1A (1), S5a (2) and PIPSL (3), and chimpanzee PIPSL (4) proteins were overexpressed in 293T cells, IP’d against HA epitope, and tested for phosphorylation of PI4P to produce PIP2. (B) The amounts of IP’d proteins were analyzed by an anti-HA Western blot. (C) Coimmunoprecipitation of HA-tagged human PIP5KA (1), S5a (2), S5AL (3), chimpanzee PIPSL (4), and untransfected 293T cells (5) with cellular ubiquitinated proteins (bracket). After anti-HA IP, proteins were immunoblotted for ubiquitin (C) or for HA-tag (D). (E) Anti-Ub Western blot of cellular lysates to demonstrate equal levels of endogenous ubiquitinated proteins. (F) Equal loading of lysates confirmed by Western against S10a. (G) Average band intensities of ubiquitinated protein from three independent co-IP experiments were normalized by HA-tagged protein amounts and scaled to the same overall blot intensity. Error bars denote ±1 SD. (*) Immunoglobulin chains; (**) a nonspecific band.