![Figure 2. Par1b promotes IRSp53 phosphorylation and 14-3-3 binding. (A) IRSp53 in total lysates from Par1b and Par1b-KD cells (1/10 input) and from lysate fractions adsorbed on immobilized 14-3-3τ and competitively eluted. Arrows show a decrease in IRSp53 mobility in the Par1b lysates. (B) In vitro kinase assays of recombinant IRSp53 with Par1b-WT or Par1b-KA followed by λ-phosphatase (+) or mock (m) treatment and subsequent binding to immobilized 14-3-3τ. (top) 1/10 of IRSp53 input is shown. (middle) IB is shown, with IRSp53 competitively eluted from 14-3-3 resin. (bottom) Autorad is shown, with [32P]γATP-Par1b–phosphorylated IRSp53. (C) IRSp53-HA or IRSp53ΔSH3-HA cells transduced with control, Par1b-WT, or Par1b-KA adenovirus. IB of 1/10 Par1b, IB of HA input, and phospho-serine after HA IP are shown. (D) IRSp53-HA immunoprecipitated from [32P]orthophosphate-labeled (autorad) and unlabeled (IB, IRSp53-HA) Par1b-KD cells. Par1b IB shows dox-dependent depletion of the two Par1b splice variants.](https://cdn.rupress.org/rup/content_public/journal/jcb/192/3/10.1083_jcb.201007002/5/jcb_201007002_gs_fig2.jpeg?Expires=1771678019&Signature=jWKAq2lLtgz5QsC8LevyHd2nupDAREmA3zdYgyR-vEpH7vI0CPsrDRqsjUuodscDpQo2LPuSVk81rpKYrOxrxNAXNvKUMtm6MNCFhqfr9yeaCuNnm3uKJxtFqkyyDoK6VkORU6jw-P5IT2~N34VYbMkLyy-ftQqPMbNCv487YKyZJE8avom9GOE1Euv1LsIqx-vYDlpurceiPsYMGXME4j2w2IyiomF-lz9aOd8e8Hj1O8FQp7xRRS-XWFL6Lg8zHNb~fdZ1oV6qoUzUjD9gCe5SiGqfZg-HntZ4Jk-9vj8hRSIyKaDUqnR4phRcgX1MNOw3j6wltkmNcEkHmZC2qQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Par1b promotes IRSp53 phosphorylation and 14-3-3 binding. (A) IRSp53 in total lysates from Par1b and Par1b-KD cells (1/10 input) and from lysate fractions adsorbed on immobilized 14-3-3τ and competitively eluted. Arrows show a decrease in IRSp53 mobility in the Par1b lysates. (B) In vitro kinase assays of recombinant IRSp53 with Par1b-WT or Par1b-KA followed by λ-phosphatase (+) or mock (m) treatment and subsequent binding to immobilized 14-3-3τ. (top) 1/10 of IRSp53 input is shown. (middle) IB is shown, with IRSp53 competitively eluted from 14-3-3 resin. (bottom) Autorad is shown, with [32P]γATP-Par1b–phosphorylated IRSp53. (C) IRSp53-HA or IRSp53ΔSH3-HA cells transduced with control, Par1b-WT, or Par1b-KA adenovirus. IB of 1/10 Par1b, IB of HA input, and phospho-serine after HA IP are shown. (D) IRSp53-HA immunoprecipitated from [32P]orthophosphate-labeled (autorad) and unlabeled (IB, IRSp53-HA) Par1b-KD cells. Par1b IB shows dox-dependent depletion of the two Par1b splice variants.
