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. 2008 Oct 23;295(6):G1246–G1254. doi: 10.1152/ajpgi.90461.2008

Fig. 5.

Fig. 5.

Coimmunoprecipitation of PKC-α with calponin in rabbit colon smooth muscle cells transfected with full-length PKC-α, various truncated forms of PKC-α in response to acetylcholine. Cells transfected with full-length PKC-α or various truncated forms of PKC-α were treated with 0.1 μM acetylcholine for 4 min. Untreated cultured cells served as controls. Cell lysate were immunoprecipitated with anti-calponin antibody and were further subjected to SDS-PAGE followed by Western blot with anti-PKC-α and anti-calponin antibodies. A: Western blot showing an increase in the association of PKC-α with calponin in cells transfected with full-length PKC-α (78 kDa) and PKC-α (C2-C3-C4) (53 kDa) in the presence of acetylcholine. Lane 1, Control (untransfected cells); lane 2, PKC-α (full-length) transfected cells; lane 3, PKC-α (full-length) transfected cells stimulated with acetylcholine; lane 4, PKC-α (C1-C2) transfected cells; lane 5, PKC-α (C1-C2) transfected cells stimulated with acetylcholine; lane 6, PKC-α (C1-C2-C3) transfected cells; lane 7, PKC-α (C1-C2-C3) transfected cells stimulated with acetylcholine; lane 8, PKC-α (C2-C3) transfected cells; lane 9, PKC-α (C2-C3) transfected cells stimulated with acetylcholine; lane 10, PKC-α (C3-C4) transfected cells; lane 11, PKC-α (C3-C4) transfected cells stimulated with acetylcholine; lane 12, PKC-α (C2-C3-C4) transfected cells; lane 13, PKC-α (C2-C3-C4) transfected cells stimulated with acetylcholine. B: stimulation of cells transfected with either full-length PKC-α or PKC-α (C2-C3-C4) with acetylcholine (0.1 μM) resulted in a significant increase in the association with calponin (123.76 ± 3.24, 120.78 ± 8.99%, respectively, for full-length and C2-C3-C4; P < 0.05, n = 3). Calponin failed to associate with truncated forms of PKC-α in cells transfected with either PKC-α (C1-C2), PKC-α (C1-C2-C3), PKC-α (C2-C3), and PKC-α (C3-C4) or upon stimulation with acetylcholine (98.28 ± 11.22, 91.16 ± 5.82, 96.63 ± 9.64, 96.19 ± 1.09%, respectively, for C1-C2, C1-C2-C3, C2-C3, and C3-C4). IB, immunoblot; IP, immunoprecipitation.