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. 2010 Jan 13;30(2):449–463. doi: 10.1523/JNEUROSCI.4992-08.2010

Figure 2.

Figure 2.

Stimulation of Pyk2 binding to PSD-95 and Pyk2 autophosphorylation in intact neurons. A, B, Acute cortical slices (A) or primary hippocampal cultures (B; 18 DIV) were incubated with TTX (1 μm) plus vehicle (Control), NMDA (50 μm), ionomycin (1 μm), or PMA (100 nm) for 15 min and extracted (1% deoxycholate) before immunoprecipitation (IP) with control IgG and then anti-PSD-95. Immunoblots (IB) were probed with anti-PSD-95 and reprobed with monoclonal Pyk2 antibody (top). Immunosignals were quantified by film densitometry (bottom). Although the variability in PSD-95 precipitation was low, Pyk2 signals were corrected by dividing each Pyk2 signal by the corresponding PSD-95 signal. The Pyk2/PSD-95 ratio was normalized to control equaling 1. Immunoblotting of lysates illustrates that the same amounts of Pyk2 were present in all samples (middle). Stimulation with NMDA, ionomycin, or PMA induced a several fold increase in Pyk2 coimmunoprecipitation with PSD-95 in slices and cultures. C, Acute cortical slices were preincubated with calmodulin inhibitors (10 μm W7, 20 μm TFP, and 30 μm calmidazolium) for 15 min before incubation with TTX plus vehicle or NMDA, processing, and analysis as in A. Control IgG precipitations (middle column) were from the same blots and exposures as PSD-95 precipitations (left column). All three calmodulin inhibitors prevented the NMDA-induced increase in coprecipitation of Pyk2 with PSD-95 (bottom row) without changing total Pyk2 in lysates (right column). D, Primary hippocampal cultures (18 DIV) were pretreated with calmodulin inhibitors before incubation with TTX plus vehicle or NMDA. Immunoblotting was performed on lysates with the phosphospecific antibody against Pyk2 Tyr402 (pY402) and, after stripping, monoclonal Pyk2 antibody. Signals were quantified by densitometry. The pY402/total Pyk2 ratio was normalized to control equaling 1. NMDA induced a several fold increase in Pyk2 Tyr402 phosphorylation, which was blocked by all three calmodulin antagonists. A–D, Asterisks indicate statistical significance compared with control (*) or NMDA (**) treatment (t test, p < 0.05; n = 4 ± SEM for all experiments).