Figure 2.
NS1 inhibits KRAS signalling in mouse and human PDAC cells. Mutant KRAS-expressing murine (KPC) and human (CFPAC) PDAC cell lines were infected with lentivirus encoding a doxycycline (DOX)-inducible NS1 expression construct to generate stable cell lines. A) Following DOX induction, NS1 was immunoprecipitated from both KPCNS1 and CFPACNS1 cells followed by Western blot with a KRAS antibody (OP24, Millipore Sigma). Panels below illustrate expression of proteins in whole cell lysates (WCL). B) Effects of DOX-induced NS1 expression on ERK and AKT activation in mouse KPCNS1 PDAC cells. ERK and AKT activation was measured by Western blot of cell lysates with phosphospecific antibodies. Vinculin was used as a normalization control for protein loading. C, D) Quantification of pERK (c) or pAKT (d) from KPCNS1 cell lysates in (b) was done using LI-COR Biosciences Image Studio Lite software (v.5.2.5) and presented as relative pERK or pAKT activation compared to no DOX lysates. Error bars represent s.d. from n = 3 independent experiments. p values were calculated by unpaired, two-tailed t test and are indicated on the graphs.