Fig. 2

miR-449a targets Jag1 by binding to its 3’UTR. a Differentiated C2C12 cells were transfected with either the scramble (Scr) or the miR-449a mimic (1–75 nM). On termination of incubation (48 h), cells were lysed and 40 μg protein was resolved on SDS-PAGE and subjected to Western blot analysis using anti-Jag1 antibody. Hsc70 was taken as the loading control. b Differentiated C2C12 cells were incubated with miR-449a mimic (10 nM) alone or together with its inhibitor (10 nM). Control cells were transfected with scramble (Scr). After 48 h, Jag1 protein levels were assessed by Western blot analysis and Hsc70 was taken as the loading control. c C2C12 cells incubated as in (b) were assessed for the transcript levels of Jag1 by qRT-PCR. 18S rRNA was used as the normalisation control. d C2C12 cells were transfected with either scramble (Scr) or the miR-449a inhibitor (10, 25 nM) and incubated for 48 h. Protein levels of Jag1 were evaluated by western blot analysis and Hsc70 was taken as loading control. e Human primary skeletal muscle cells were transfected with either the scramble (Scr) or the miR-449a mimic (10 nM) alone or with its inhibitor and western blot analysis was performed using anti-Jag1 antibody. HSC70 was used as the loading control. f C2C12 cells were plated in 12-well plates and transfected with the wild-type (WT) or the mutated (mut) Jag1 3′ UTR (100 ng) together with the miR-449a mimic (10 nM) and/or its inhibitor (10 nM). Control cells were transfected with the scramble (Scr). After 48 h, cells were lysed and luciferase activity was measured as described in the ‘Materials and methods’. Firefly luciferase values were normalised to the values of Renilla luciferase. g The protein expression of Jag1 was assessed in db/+ and db/db mice by western blot analysis. Briefly, 40 μg protein from skeletal muscle of db/+ and db/db mice were separated on SDS-PAGE, transferred to nitro-cellulose membranes and probed with anti-Jag1 antibody. Hsc70 was used as the loading control. Densitometric analysis is given along with the respective blots. All experiments were done at least thrice and values present are mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05