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. 2012 May 18;7(5):e34782. doi: 10.1371/journal.pone.0034782

Figure 3. gAd activates a mesenchymal motility program in mSAT.

Figure 3

mSAT were treated overnight with free-serum medium and then stimulated with gAd (1 µg/ml) for the period indicated. A) Rac-1 activation assay. 50 µg of total proteins were used in the GST pull down assay for Rac-1 and the amount of GTP-bound Rac-1 was detected by anti-Rac-1 immunoblot after the pull down. Normalization has been carried out by anti-Rac-1 immunoblot analysis of cell lysates. Normalized data are shown in the bar graph. *p<0,001 vs time 0. B) Expression of mesenchimal markers Twist, Snail and vimentin. 20 ug of total proteins were loaded in each lane and the expression level of Snail-1, Twist and vimentin was analysed by immunoblots. Anti-actin immunoblot was used for normalization and the normalized data are reported in the bar graph, *p<0,001 vs time 0. C), D) mSAT were permanently transduced with AdipoR1 (C) or AdipoR2 (D) shRNAs and treated as described in B), *p<0,01 and #p<0,025 vs time 0. E) Expression analysis of AdipoR1 and AdipoR2 in mSAT after transduction with AdipoR1 or AdipoR2 shRNAs. All immunoblots are representative of three different and independent experiments.