Fig. 2

Increased PINCH expression disrupts the PIP complex and is accompanied by actin disassembly. a Cell lysates from control, Tat- or TNFα-treated neurons were immunoprecipitated with anti-PINCH antibody. Following immunoprecipitation, total cell lysates (input) and immunoprecipitated materials (IP) were subjected to Western blot analysis. Samples were probed with antibodies specific for PINCH (top left), ILK1 (top right), α-Parvin (bottom left), and TESK1 (bottom right). b Changes in cellular actin cytoarchitecture were observed using confocal microscopy in neurons untreated or exposed to Tat or TNFα. Neurons were fixed, permeabilized and labeled with anti-PINCH and stained with phalloidin, the dye that stains actin filaments. Representative confocal images show depolymerization of actin in human neurons exposed to Tat/TNFα. c and d Quantification of the PINCH fluorescence (c) and the actin filament length (d). e Representative Western blot for lysates from neurons untreated or exposed to Tat or TNFα and probed with antibodies against phospho-Cofilin, Cofilin, Chronophin, and GAPDH. f and g Quantification of relative protein abundance of phospho-Cofilin/Cofilin (f) and Chronophin/GAPDH (g) from (e). h Cell lysates from control, Tat- or TNFα-treated neurons were immunoprecipitated with anti-Parvin antibody. Following immunoprecipitation, total cell lysates (input) and immunoprecipitated materials (IP) were subjected to Western analysis. Samples were probed with antibodies specific for Parvin, TESK1, and ILK1. i Schematic representation of PIP complex regulation of cofilin phosphorylation and actin depolymerization. Data represent mean ± SEM; **P < 0.01; ***P < 0.001; n = 3–5 (one-way ANOVA)