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Bacterial Expression System> | The coding sequences of human PI3K p85α (catalog no. OHu21143D; GenScript) corresponding to 1–315 aa and various human CD2AP constructs (FL-CD2AP 1–639 aa; CD2AP 1–335 aa, 1–475 aa, 336–605 aa; GFP-1-475 aa; and GFP-1- 335 aa) were amplified by PCR (iProof) and cloned in-frame into the bacterial expression vector pET30a+ (EMD) with 6-His tag at the N-terminus for expression in BL21 cells (NEB). | Get A Quote |
Maintaining the correct ratio of apical, basal, and lateral membrane domains is important for epithelial physiology. Here, we show that CD2AP is a critical determinant of epithelial membrane proportions. Depletion of CD2AP or phosphoinositide 3-kinase (PI3K) inhibition results in loss of F-actin and expansion of apical-basal domains, which comes at the expense of lateral membrane height in MDCK cells. We demonstrate that the SH3 domains of CD2AP bind to PI3K and are necessary for PI3K activity along lateral membranes and constraining cell area. Tethering the SH3 domains of CD2AP or p110γ to the membrane is sufficient to rescue CD2AP-knockdown phenotypes. CD2AP and PI3K are both upstream and downstream of actin... More