Pre-stained protein markers (10?50 kDa; Bio-Rad, Precision Plus: Dual colour) were used as molecular weight markers on each gel. In order to control for between gel variations, an internal standard made of pooled cerebellar samples from sham rats was loaded on each gel. The samples were electrophoresed with a 90 V variable current (BioRad, PowerPack 3000) until protein flattened at the stacking/resolving interface, and 180 V thereafter. The proteins were transferred to polyvinylidene-difluoride (PVDF) membranes using a transblotting apparatus (2.5 L; Bio-Rad). The transfer was performed overnight in transfer buffer (25 methanol, 1.5 glycine and 0.3 Tris-base) at a 10 V variable current (Bio-Rad PowerPack 3000). Non-specific IgG binding was blocked by incubation with 5 dried milk protein (Pams) and 0.1 bovine serum albumin (BSA) (Sigma) for 6? h at 4uC. The membranes were then incubated with affinity-purified polyclonal goat antibodies raised against GluR1, GluR2, GluR3 and GluR 4, and affinity-purified polyclonal rabbit antibodies raised against NMDA e1 (NR1), NMDA e2 (NR2A), NMDA f1 (NR2B), CaMKIIa and pCaMKIIa, overnight at 4uC (see antibody details in Table 1). The specificity of these antibodies has been demonstrated in previous studies (NR1 [31]; NR2A [32]; NR2B [33]; GluR1 [34]; GluR2 [35]; GluR3 [36]; GluR4 [37]; CaMKIIa [38]; pCaMKIIa [39]; b-actin [40]) and the LY2409021 chemical information dilutions were optimised for the current study. The secondary antibodies were anti-goat IgG linked to horseradish peroxidase and antirabbit IgG linked to horseradish peroxidase (see details in Table 1). Detection was performed using the enhanced chemiluminescence (ECL) system (Amersham Biosciences, NZ). Hyperfilms (Amersham Biosciences, NZ) were analyzed by densitometry to determine the quantity of protein expressed in each group usingGlutamate Receptors after Vestibular DamageFigure 3. Example of western blots for CamKIIa and pCaMKIIa in CA2/3 for the BVD (`B’) and sham (`S’) animals that received T maze training or no T maze training at 6 months post-op. `IS’ is the internal standard and b-actin (`Actin’) is also shown. doi:10.1371/journal.pone.0054527.ga calibrated imaging densitometer (Bio-Rad) and a PowerPC Mac running OS 9.2 and Quantity One software. Results were expressed as the volume of the band, i.e., optical density 6area of the band. An antibody against b-actin (see details in Table 1) was used as a loading control and exploratory regression analyses performed in our laboratory have shown that any changes in b-actin expression were unlikely to account for changes in the target protein expression (R2 = 0.087) [30]. The volume of each target band was then normalised to its corresponding loading control and then the internal standard within each gel. It was expected that the protein levels measured would reflect both the intra-cytoplasmic and membrane receptor subunits together.performed a series of further multivariate statistical analyses. POR 8 Linear discriminant analysis (LDA) was performed, with Wilks’ l as the test statistic and leave one out cross-validation [41,43]. Finally, cluster analyses were performed on the data expressed as z scores using Ward’s minimal variance algorithm and the correlation coefficient distance [41,42]. The significance level was set at 0.05 for all comparisons.ResultsBVD resulted in a number of postural and locomotor behavioural symptoms 26001275 that are characteristic of bilateral vestibular loss. These included: gait ataxia, marked.Pre-stained protein markers (10?50 kDa; Bio-Rad, Precision Plus: Dual colour) were used as molecular weight markers on each gel. In order to control for between gel variations, an internal standard made of pooled cerebellar samples from sham rats was loaded on each gel. The samples were electrophoresed with a 90 V variable current (BioRad, PowerPack 3000) until protein flattened at the stacking/resolving interface, and 180 V thereafter. The proteins were transferred to polyvinylidene-difluoride (PVDF) membranes using a transblotting apparatus (2.5 L; Bio-Rad). The transfer was performed overnight in transfer buffer (25 methanol, 1.5 glycine and 0.3 Tris-base) at a 10 V variable current (Bio-Rad PowerPack 3000). Non-specific IgG binding was blocked by incubation with 5 dried milk protein (Pams) and 0.1 bovine serum albumin (BSA) (Sigma) for 6? h at 4uC. The membranes were then incubated with affinity-purified polyclonal goat antibodies raised against GluR1, GluR2, GluR3 and GluR 4, and affinity-purified polyclonal rabbit antibodies raised against NMDA e1 (NR1), NMDA e2 (NR2A), NMDA f1 (NR2B), CaMKIIa and pCaMKIIa, overnight at 4uC (see antibody details in Table 1). The specificity of these antibodies has been demonstrated in previous studies (NR1 [31]; NR2A [32]; NR2B [33]; GluR1 [34]; GluR2 [35]; GluR3 [36]; GluR4 [37]; CaMKIIa [38]; pCaMKIIa [39]; b-actin [40]) and the dilutions were optimised for the current study. The secondary antibodies were anti-goat IgG linked to horseradish peroxidase and antirabbit IgG linked to horseradish peroxidase (see details in Table 1). Detection was performed using the enhanced chemiluminescence (ECL) system (Amersham Biosciences, NZ). Hyperfilms (Amersham Biosciences, NZ) were analyzed by densitometry to determine the quantity of protein expressed in each group usingGlutamate Receptors after Vestibular DamageFigure 3. Example of western blots for CamKIIa and pCaMKIIa in CA2/3 for the BVD (`B’) and sham (`S’) animals that received T maze training or no T maze training at 6 months post-op. `IS’ is the internal standard and b-actin (`Actin’) is also shown. doi:10.1371/journal.pone.0054527.ga calibrated imaging densitometer (Bio-Rad) and a PowerPC Mac running OS 9.2 and Quantity One software. Results were expressed as the volume of the band, i.e., optical density 6area of the band. An antibody against b-actin (see details in Table 1) was used as a loading control and exploratory regression analyses performed in our laboratory have shown that any changes in b-actin expression were unlikely to account for changes in the target protein expression (R2 = 0.087) [30]. The volume of each target band was then normalised to its corresponding loading control and then the internal standard within each gel. It was expected that the protein levels measured would reflect both the intra-cytoplasmic and membrane receptor subunits together.performed a series of further multivariate statistical analyses. Linear discriminant analysis (LDA) was performed, with Wilks’ l as the test statistic and leave one out cross-validation [41,43]. Finally, cluster analyses were performed on the data expressed as z scores using Ward’s minimal variance algorithm and the correlation coefficient distance [41,42]. The significance level was set at 0.05 for all comparisons.ResultsBVD resulted in a number of postural and locomotor behavioural symptoms 26001275 that are characteristic of bilateral vestibular loss. These included: gait ataxia, marked.
