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. Author manuscript; available in PMC: 2010 Oct 19.
Published in final edited form as: Cancer Cell. 2009 Aug 4;16(2):115–125. doi: 10.1016/j.ccr.2009.06.006

Figure 7. Knocking down the expression of INPP4B increases the duration of AKT activation in response to insulin.

Figure 7

(A) Stable HMEC cell pools expressing shRNA vectors directed against Renilla, PTEN, INPP4B (‘INPP4B-1’) or HMEC cell pools expressing the knockdown resistant INPP4B expression construct in the shRNA-INPP4B-1 cell pool (‘INPP4B-1 + INPP4BR’) were serum-starved and stimulated with 100nM insulin for the indicated time periods. Cell lysates were separated by SDS-PAGE and phospho-Akt detected with specific antibodies in Western blot analysis. INPP4B knockdown cell pools display increased and prolonged phospho-Thr308 and phospho-Ser473 Akt compared to Renilla control knockdown cell pools. Expression of a Flag-tagged INPP4B construct harboring two silent mutations at Ser487 to render it resistant to shRNA knockdown in shRNA-INPP4B-1 cell pools (INPP4BR) reversed prolonged Akt phosphorylation. The results are representative of 4 separate experiments.

(B) Quantification of phospho-Thr308 Akt normalized to total Akt levels (n = 4, *p< 0.1, **p<0.01, ***p<0.001). Quantification of each single time point was determined using NIH ImageJ (n = 4). Data are shown as mean +/− SEM.