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. 2012 Jul 25;32(30):10365–10369. doi: 10.1523/JNEUROSCI.0851-11.2012

Figure 1.

Figure 1.

Alternatively spliced exons e37a and e37b influence ubiquitination of CaV2.2 channels. A, Left, Pattern of mutually exclusive alternative splicing of e37a and e37b in the CaV2.2 encoding gene, Cacna1b. Right, Amino acid sequences of e37a and e37b aligned; asterisks denote 19 aa conserved between exons. Arrow highlights critical Y1747 in e37a and F1747 in e37b. B–D, Western blots compare levels of ubiquitin associated with e37a-CaV2.2 and 37b-CaV2.2 protein isolated from tsA201 cells. Cells were transfected with cDNAs for HA-Ub, CaVβ3, and e37a-CaV2.2 (37a) or e37b-CaV2.2 (37b). B, Protein immunoprecipitated with antibodies to CaV2.2, same membrane probed with anti-HA-Ub (top) then sequentially stripped and reprobed with anti-Ub (middle), and anti-CaV2.2 (bottom). Black bar is the 250 kDa marker. C, Relative signal intensities for e37a (top) and e37b (bottom) signals plotted according to migration into the gel and referenced to the 250 kDa marker. Image density analyzed with ImageJ software. Signal intensities were normalized to the peak of e37a signal for comparison. D, Western blots of protein lysate (left) and immunoprecipitated using CaV2.2 antibody (right) from tsA201 cells transfected with e37a-CaV2.2, e37b-CaV2.2, or empty vector (−). Membrane probed with anti-HA-Ub (top) and stripped and reprobed with anti-CaV2.2 (bottom) illustrates the specificity of both HA-Ub and CaV2.2 antibodies.