Fig 1: Mimicking Ser71 phosphorylation by the S71D mutation abolishes actin-binding of Pfn1. (A) X-ray diffraction structure of human Pfn1 (right, cyan) bound with actin (left, purple) (Protein Data Bank ID 2PAV) (Ferron et al., 2007). Ser71 and four other serine and threonine residues in the actin-binding region of Pfn1 are displayed in the sticks mode. (B) HEK293T cells were transfected with Myc-tagged wild type or mutant Pfn1 containing alanine (A) or aspartate (D) substitutions on the five candidate serine or threonine residues. Lysates were subjected to immunoprecipitation using a Myc-tag antibody, and Western blot was used to detect Myc-Pfn1 and co-precipitated actin. (C) HEK293T cells were transfected with wild type or mutant forms of HA-tagged Pfn1 containing S71A or S71D, which were subsequently immunoprecipitated by an anti-HA tag antibody. Lysates were subjected to immunoprecipitation, and samples were blotted for actin, VASP, and HA-tag (to visualize HA-Pfn1).