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. 2014 Jun 23;205(6):829–846. doi: 10.1083/jcb.201403021

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© 2014 Taylor et al.

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Figure 9. — Ca_vβ_1a action at the Myog promoter. (A) Myog-luc expression in GFP (black) or Ca_vβ_1a-YFP (green) expressing myoblasts (n = 5) and myotubes (n = 6). (B) Myog-luc expression in control (black) and Ca_vβ_1a-shRNA (red)–transfected C2C12 myoblasts (n = 3). (C) ChIP-qPCR showing relative fold enrichment of Ca_vβ_1a-YFP pull-down of Myog promoter (Mgn 5′) and Tnnt3 promoter (Tnnt3), with Myog 3′ region (Mgn 3′) and Cacnb1 exon 5 (Cacnb1) as controls. (D) Gel shift assay using GFP protein (control) or Ca_vβ_1a-YFP protein from Cos7 nuclear extracts. Mouse IgG is nonspecific antibody. A specific shift can be seen in lanes 2, 7, and 10 (white carats), and supershift induced by YFP antibody seen in lane 11 (black arrowheads). Fluorescently labeled probe sequences (top) were generated from ChIP-chip motif results (sequences #1 and #2) and from the Myog promoter (sequence #3; NC E-box motif underlined). Full probe sequences are available in Table S1. (E) Mutation analysis of Myog promoter. C2C12 were transfected with GFP (black) or Ca_vβ_1a-YFP (green) and then wild-type (−184 +48) Myog-luc, or −123 +48 fragments with mutations in E1 E-box (ΔE1), Pbx1 (ΔPbx), or noncanonical E-box (ΔNC E-box; CAGCTTA sequence indicated in D has been mutated to TGGCTTA) Myog-luc constructs, n = 3 per group. Locations of mutations are indicated above. See Berkes et al. (2004) for origin of these constructs. Data are ± SEM; *, P ≤ 0.05; ***, P ≤ 0.001.