The Journal of Neuroscience, March 8, 2006, ():
Alternative Splicing of the Voltage-Gated Ca2+ Channel 
4 Subunit Creates a Uniquely Folded N-Terminal Protein Binding Domain with Cell-Specific Expression in the Cerebellar Cortex
J. Neurosci. Vendel et al.
26: 2635
Supplemental data
Files in this Data Supplement:
- supplemental material
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Figure S1. Testing for specificity of β subunit antibodies using purified recombinant A-domain proteins. A, Specificity of anti-β4a-A and anti-β4b-A affinity-purified polyclonal antibodies. Purified recombinant β4a-A and β4b-A were subjected to SDS-PAGE and either stained with Coomassie blue (Lanes 1 and 2 from left to right) or transferred to a nylon membrane for Western blot analysis (Lanes 3-6). Coomassie staining reveals that approximately equal amounts of β4a-A and β4b-A protein were loaded on to the gel. Western blot analysis demonstrates that the anti-β4a-A antibody (1:5000) labels only purified β4a-A protein (Lanes 3 and 4) and that the anti-β4b-A antibody (1:1000) labels only the β4b-A protein. Staining below each of the prominently labeled bands is indicative of some degradation of purified protein. B, The anti-β4a-A antibody does not cross-react with the 6His-β3 A-domain. Purified recombinant β4a-A and 6His-β3-A were subjected to SDS-PAGE and either stained with Coomassie blue (Lanes 1 and 2 from left to right) or transferred to a nylon membrane for Western blot analysis (Lanes 3-9). Coomassie staining reveals that approximately equal amounts of β4a-A and 6His-β3-A protein were present in the 1X stock solutions used to load the gel. Western blot analysis demonstrates that the anti-β4a-A antibody (1:1000) labels purified β4a-A protein (0.1X protein relative to stock solution, Lane 3), but does react with β3-A protein loaded on to the gel at 0.1X, 0.5X, and 1X concentrations relative to the stock solution (Lanes 4-6, respectively). Western blot analysis using an anti-6His antibody (1:1000) demonstrates that the 6His-β3-A protein was successfully transferred to the nylon membrane, with staining evident at 0.5X and 1X concentrations of 6His-β3-A (Lanes 7-9). Coomassie staining of the polyacrylamide gel after protein transfer verified that both β4a-A and 6His-β3-A proteins transferred with equal efficacy (data not shown). Numbers to the left of each panel indicate molecular mass in kDa.
- supplemental material
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Figure S2. Specificity of β subunit antibody labeling in cerebellar cortex. As a partial test to determine whether β subunit antibody labeling in cerebellar cortex is due to specific binding to β subunit A-domains, anti-β4a-A and anti-β4b-A affinity-purified polyclonal antibodies were pre-incubated with purified recombinant β4a-A and β4b-A protein (1:1 mass ratio) prior to standard immunolabeling procedures. Anti-β3 antibody was pre-incubated with the β3 subunit C-terminal peptide antigen that was included with the purchase of the antibody (Chemicon). In each case (β4a, top two panels; β4b, middle two panels; and β3, lower two panels), pre-absorption with protein antigen eliminated specific labeling; however, as can be seen in the right-hand panels, light shadows of Purkinje cell bodies were still visible.
