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. 2017 Jun 28;7(6):170024. doi: 10.1098/rsob.170024

Figure 7.

Figure 7.

Experimental repression of Ubc9 in primary and cultured epithelial cells downmodulates active Akt1. (a) Immunoblots with indicated antibodies from lysates of HCT-8 cells with either Akt1 knockdown using Akt1-siRNA (AKD), Ubc9 knock-down using Ubc9-siRNA (UKD), Ubc9 over-expression using pUbc9 (UOE), along with corresponding control lysates (Ctl). Corresponding densitometric values of total Akt1 (b), ratios of pAkt1/Akt1 (c) and Ubc9 (d) were also represented as bar graphs. (e) Immunoblots of Ubc9 from HCT-8 cells treated specifically with Akt1 kinase inhibitor (AKTinh), siRNA-for Akt1 (AKD), UKD or left untreated (Ctl). (f) Schematic overview of steps involved in ex vivo culturing of PIECs from mice colon (actual picture of epithelial culture represented here). (g) Immunoblots of Ubc9, p-Akt1 and total Akt1 from lysates of PIEC treated with the indicated inhibitors (AKTinh, Akt1 inhibitor; LLO, listeriolysin; SAM, S-adenosyl methionine). Right panel: densitometric analysis of immunoblots. (h,i) ELISA for IFN-γ and IL-10 of supernatant of PIECs treated as indicated. Supernatant of DSS-7 mice primary epithelial cells were used as positive controls for inflammation.