Fig 1: TNFa increases SRp40 phosphorylation through AKT: (A) Representative autoradiograph of Western blot analysis (left panel) of SRp40 phosphorylation (i.e., pSRp40) levels in hMSCSc at T = 0 h treated for 30 min with 10 or 20 ng/mL of TNFa cytokine or with vehicle (i.e., PBS). Hsp90 was used as a loading control. Bar graphs (right panel) report relative pSRp40 levels normalized on Hsp90 expression (pixel density analysis of Western blots). Data are representative of the mean ± SEM of three independent experiments. *** p = 0.001. (B) Representative Western blot of pSRp40 (left panel) on lysates from undifferentiated hMSCs treated for 24 h with conditioned medium (CM) of LPS-activated MF (THP-1) or control medium plus 0.5 mg/mL of human neutralizing antibody against TNFa or anti-IgG1 Isotype. Hsp90 was used as loading control. Bar graphs (right panel) report relative pSRp40 levels normalized on Hsp90 expression (pixel density analysis of Western blots). Data are representative of the mean ±SEM of three independent experiments. ** p = 0.01. (C) Representative immunoblotting of Akt phosphorylation (i.e., pAkt) and total Akt levels (right panel) in hMSCSc at T = 0 h treated for 30 min with 10 or 20 ng/mL of TNFa cytokine or with vehicle (i.e., PBS). Hsp90 was used as loading control. Bar graphs (right panel) report relative pAkt levels normalized on Hsp90 and total Akt expression (pixel density analysis of Western blots). Data are representative of the mean ± SEM of three independent experiments. (D) Representative autoradiograph of Western blot analysis (left panel) of pAkt and total Akt levels in undifferentiated hMScs treated for 24 h with the CM of LPS-activated MF (THP-1) or control medium plus 0.5 mg/mL of human neutralizing antibody against TNFa or anti-IgG1 Isotype. Hsp90 was used as loading control. Bar graphs (right panel) report relative pAkt levels normalized on Hsp90 and total Akt expression (pixel density analysis of Western blots). Data are representative of the mean ± SEM of at least three independent experiments. * p = 0.05 and ** p = 0.01. (E) Representative immunoblotting of pSRp40 (left panel) in undifferentiated hMSCs treated with Wortmannin or vehicle (i.e., DMSO) in combination and not with 10 or 20 ng/mL of TNFa cytokine. Hsp90 was used as loading control. Bar graphs (right panel) report relative pSRp40 levels normalized on Hsp90 expression (pixel density analysis of Western blots). Data are representative of the mean ± SEM of at least three independent experiments. * p = 0.05 and ** p = 0.01.