Figure 7. BVR bridges FAK to Src to mediate synaptic focal adhesion signaling.

(A) Schematic depicting strategy for inserting 3xFLAG tag into the endogenous mouse Blvra locus. (B) Immunoblots of brain lysates from Blvra^FLAG and 3 Blvra^WT mice for FLAG and BVR. β-actin serves as loading control. (C) Immunoblot of 3xFLAG-BVR in various subcellular fractions of brain tissue from Blvra^FLAG mice. H2B and GluA1 immunoblots were blotted to serve as markers of nuclear and synaptosomal fractions, respectively. (D and E) Immunoblots of (D) FAK, Pyk2, Src and FLAG-BVR and (E) NR2A, NR2B, PSD-95, and FLAG-BVR in lysates and anti-FLAG immunoprecipitates (IP) from Blvra^WT and Blvra^FLAG mouse brain tissue. (F and G) Immunoblots and quantification of lysates following anti-FLAG IP from WT and BVR^−/− MEFs overexpressing V5-Pyk2 and FLAG-Src. Data are expressed as normalized ratio of V5-Pyk2 with anti-FLAG IP to FLAG-Src with anti-FLAG IP. V5 input serves as loading control. (H to M) Immunoblots and quantification of lysates following anti-Src IP from WT and BVR^−/− hippocampi. Data are expressed as the ratios of (I) FAK, (J) Pyk2, (K) NR2A, (L) NR2B, and (m) PSD-95 that immunoprecipitated with Src, normalized to their respective expression in the input. (N to P) Immunoblots and quantification of lysates and GST IP from HEK 293 overexpressing GST-BVR, myc-FAK, and FLAG-Src treated with vehicle or 500 nM PMA for 30 min. Data are expressed as the ratios of (O) myc-FAK or (P) FLAG-Src that immunoprecipitated with GST-BVR, normalized to their respective expression in the input. (Q and R) Immunoblots and quantification of phospho- and total ectopically expressed NR2B in WT and BVR^−/− MEFs treated with vehicle or 500 nM PMA for 30 min. All data are mean ± SEM depicted from independent experiments. ^ns P > 0.05 and ** P < 0.01 by two-tailed unpaired Student’s t-test.