Detection of JAK2V617F protein levels and activities in transgenic mice. (A) Peripheral blood white cells from line A and B transgenic mice and their nontransgenic siblings were attached to polylysine-coated coverslips, fixed with formaldehyde, and then stained with an anti-JAK2 antibody (Cell Signaling Technology, Danvers, MA) and further probed with a Cy3-conjugated antirabbit secondary antibody. Photos were taken with a 40× objective. The arrowheads point to positively stained cells. (B) Cell extracts from bone marrow (after red cell lysis) of 2 nontransgenic control and 2 line A transgenic mice were analyzed for JAK2 tyrosine kinase activity with GST-JAKS as a substrate as described earlier.25 Detections were made using an antiphosphotyrosine antibody. Equal protein loadings are indicated by 2 irrelevant (nonspecific protein) bands recognized by the antibody. (C) Analyses of ERK activation in bone marrow cells from 2 nontransgenic control and 2 line A transgenic mice with anti-pERK antibody (Cell Signaling Technology). Coomassie blue staining of the PVDF membrane revealed equal amounts of actin, indicating equal protein loadings. Note that actin and ERK2 have similar molecular size.
