Figure 6.

Cell-dissociation enzymes rapidly disrupt intracellular precursors of Notch2 signaling and induce Notch target genes in KP1 cells. A and B, Immunoblots of full-length Notch2 and the processed Notch transmembrane (NTM) subunit in KP1 cells after treatment of 0.05% trypsin or 1x accutase for five minutes. Vinculin, tubulin, and GAPDH were used as loading controls. C, Relative abundance of total Notch2 (full-length + NTM) for the indicated conditions. Data are normalized to control KP1 cells lysed without dissociation. D, Ratiometric abundance of NTM / full-length (FL) Notch2 for the indicated conditions. E–G, Quantitative PCR of the Notch target genes Hes1 (E) and Hey1 (F) and the housekeeping gene Hprt (G) in KP1 cells one hour after treatment of 0.05% trypsin or 1x accutase for five minutes. Samples were normalized to Rpl30 and Ppia as loading controls. For C and D, data are shown as the mean ± s.e.m. from n = 4 independent biological samples. For E–G, data are shown as the geometric mean ± log-transformed s.e. from n = 4 independent biological samples. Differences in means were assessed by ANOVA with Tukey HSD post-hoc test (C and D) or log-transformed Student’s t test with Šidák correction (E–G).