I-mf, a Novel Myogenic Repressor, Interacts with Members of the MyoD Family  C.-M.Amy Chen, Norbert Kraut, Mark Groudine, Harold Weintraub  Cell  Volume 86, Issue 5, Pages 731-741 (September 1996) DOI: 10.1016/S0092-8674(00)80148-8

Figure 1 The Predicted Amino Acid Sequence of I-mf Proteins Three polypeptides, I-mfa, I-mfb, and I-mfc, are encoded by the I-mf gene. Dashes indicate that the amino acid at that position is identical to I-mfa. Cell 1996 86, 731-741DOI: (10.1016/S0092-8674(00)80148-8)

Figure 2 In Vitro and In Vivo Binding of I-mf Proteins to the MyoD Family Members (A) In vitro–translated and [35S]methionine–labeled Myf5 (lanes 1 and 2), Myogenin (lane 3), MyoD (lane 4), and E12 (lane 5) were directly analyzed by SDS-polyacrylamide gel electrophoresis (bottom panel) or analyzed after precipitation with amylose resin following incubation with purified MBP–I-mfc (lane 1) and MBP–I-mfa (lanes 2–5) fusion proteins (upper panel). (B) Cell extracts were made from NIH3T3 cells transiently transfected with expression vectors encoding Myf5 (lane 1) or MyoD (lanes 2–4) and HA epitope-tagged I-mfa (lanes 1 and 2), I-mfb (lane 3), or I-mfc (lane 4). Western blot analyses were performed on those extracts with MyoD monoclonal antibody, 5.8A (bottom panel), or anti-HA epitope antibody, 12CA5 (middle panel). The same extracts were also immunoprecipitated with MyoD antiserum and protein A beads followed by detection of precipitates with anti-HA antibody, 12CA5, by Western blot analysis (upper panel). Cell 1996 86, 731-741DOI: (10.1016/S0092-8674(00)80148-8)

Figure 3 Expression Pattern of the I-mf Gene In situ hybridization with digoxygenin-labeled antisense riboprobes derived from I-mfa (A) and MyoD (B) cDNA or with 35S-labeled antisense riboprobes derived from I-mfa (C) and Myf5 (D) cDNA were performed on adjacent parasagittal sections (A and B) of E11.5 mouse embryos or adjacent transverse sections of the caudal region of E11.5 mouse embryos. D, dermomyotome; DA, dorsal aorta; DG, dorsal root ganglion; H, heart; L, liver; N, neural tube; Sc, sclerotome; St, stomach. Cell 1996 86, 731-741DOI: (10.1016/S0092-8674(00)80148-8)

Figure 4 The Effect of I-mf Proteins on Myf5-Mediated Myogenesis A Myf5 expression vector was transiently transfected into NIH3T3 cells together with the control expression vector (A–C), the same expression vector encoding I-mfa (D–F), I-mfb (G–I), or I-mfc (J–L). Twenty-four hours after transfection, cells were grown in differentiation medium for an additional 40 hr before staining. Double immunofluorescent staining was performed with a Myf5 antiserum (A, D, G, and J) and the anti-myosin heavy chain (MyHC) monoclonal antibody, MF20 (B, E, H, and K), followed by incubation with Fluorescein-conjugated anti-rabbit Ig antibody and Rhodamine-conjugated anti-mouse Ig antibody, respectively. Superimposed images of Myf5 and MyHC staining are shown in (C), (F), (I), and (L). Cell 1996 86, 731-741DOI: (10.1016/S0092-8674(00)80148-8)

Figure 5 Subcellular Localization of Myf5 and I-mf Proteins Myf5 (A–I) or NLSMyf5 (J–L) expression vectors were transiently transfected into NIH3T3 cells together with expression vectors encoding HA epitope-tagged I-mfa (A–C, J–L), I-mfb (D–F), or I-mfc (G–I). Forty hours after transfection, cells were double immunostained with Fluorescein-conjugated anti-rabbit Ig antibody following Myf5 antiserum staining (A, D, G, and J), and Rhodamine-conjugated anti-mouse Ig antibody following staining with anti-HA epitope monoclonal antibody, 12CA5 (B, E, H, and K). Images of Myf5 and HA-I-mf staining were superimposed in (C), (F), (I), and (L). Based on the intensity of staining, Myf5 was expressed at comparable levels in cells cotransfected either with control vector, I-mfa, I-mfb, or I-mfc. Cell 1996 86, 731-741DOI: (10.1016/S0092-8674(00)80148-8)

Figure 7 The Effect of I-mf proteins on DNA-Binding Activities of the MyoD Family (A) In vitro translated Myogenin (lanes 2–10) was incubated with either no protein (lane 2), increasing molar ratios (by a factor of 2) of purified MBP–I-mfaΔN proteins (from a 1.5-fold to a 48-fold molar excess, from lane 3 to lane 8), or a 48-fold molar excess of MBP–I-mfbΔN and MBP–I-mfc (lanes 9 and 10, respectively). In vitro translated E47N (Sun and Baltimore 1991) was also incubated with either no protein or with a 192-fold molar excess of MBP–I-mfaΔN, MBP–I-mfbΔN, or MBP–I-mfc (lane 11 to 14). An electrophoretic mobility shift assay was then performed after adding the labeled B1/B2 probe. (B) In vitro translated Myogenin (lane 1–4), MyoD (lane 5–8), Myf5 (lane 9–12), and NLSMyf5 (lane 13–16) were incubated with in vitro–translated E12 together with no MBP fusion protein (lanes 1, 5, 9, and 13) or an increasing molar excess (by a factor of 2) of bacterially purified MBP–I-mfaΔN from a 48- to 192-fold molar excess relative to Myogenin (lanes 2–4) or from a 160- to 640-fold molar excess relative to MyoD, Myf5, and NLSMyf5 (lane 6–8, 10–12, and 14–16, respectively). Gel electrophoresis was conducted after addition of the labeled B1/B2 probe. Asterisk indicates the heterodimer complex. Cell 1996 86, 731-741DOI: (10.1016/S0092-8674(00)80148-8)

Figure 6 The Effect of I-mf proteins on NLSMyf5 Mediated-Muscle Differentiation A NLSMyf5 expression vector was cotransfected into NIH3T3 cells with expression vectors encoding HA epitope (A–C), HA epitope-tagged I-mfa (D–F), or HA epitope-tagged I-mfc (G–I). Twenty-four hours posttransfection, cells were grown in differentiation medium for an additional 40 hr before they were stained with antibodies. Double immunostaining was performed with Myf5 antiserum (A, D, and G) and anti-MyHC monoclonal antibody, MF20, (B, E, and H) followed by staining with Fluorescein-conjugated anti-rabbit Ig antibody and Rhodamine-conjugated anti-mouse Ig antibody, respectively. Superimposed images of Myf5 and MyHC staining are shown in (C), (F), and (I). Cell 1996 86, 731-741DOI: (10.1016/S0092-8674(00)80148-8)