Fig 2: Immunodetection of Abl/BcrAbl, CrkL, and relevant site-specific phosphorylation.Lysates from parental (K), K562-IR (I), K562-NR (N) and K562-DR (D) cells cultured in the absence (K) or presence (I, N and D) of maintenance concentrations of their respective TKIs were analysed in duplicate using the Wes capillary immunodetection system from ProteinSimple. (A) Individual capillary plots and pseudo-“blot” views (in which signal peaks are represented in “band” form) from chemiluminescence for one replicate from each antibody analysis: total Abl/BcrAbl (aAbl #2862, Cell Signaling Technology), phospho-Abl/BcrAbl (apY245 ab195839, Abcam), total CrkL (aCrkL 32H4/#3182, Cell Signaling Technology), phospho-CrkL (apY207 EP270Y/ab52908), and GAPDH (aGAPDH D16H11, Cell Signaling Technology). Peaks are labelled for the respective proteins; note that for GAPDH, one capillary lane (labelled with *) in this replicate exhibited artifactually shifted migration, however immunodetection was consistent with other capillaries. (B) Peaks from total BcrAbl, pYBcrAbl, total CrkL and pYCrkL were normalized to peaks from GAPDH for each duplicate, and plotted to show comparisons between the parental (K) and drug-resistant (I, N, D) cells. Error bars indicate SEM for duplicate runs. (C) GAPDH-normalized signals were used to calculate and compare %phosphorylated/total signal for BcrAbl (top) and CrkL (middle). Estimated % phosphorylated biosensor calibrated from the standard curve and compared with total binding capacity of the well, as described in Fig 6 legend) was re-plotted from data shown in Figs 4 and 6, and shown here (bottom) for comparison to endogenous protein %phospho/total. Note that “blot” images are digital representations of peak intensity, not photographic or scanned images of actual blots, and thus band shapes appear unnatural.

Fig 3: SNIPER(ABL)-39 inhibits the BCR-ABL-related signaling pathway. K562 cells were incubated with the indicated concentration of SNIPER(ABL)-39 or dasatinib for 6 h. pBCR-ABL, pSTAT5 and pCrkL stand for phosphorylated BCR-ABL, STAT5 and CrkL, respectively.

Fig 4: Arg promotes inflammatory vascular leak in vivo. a Quantification of extravasated Evans Blue in the lungs of untreated and LPS-treated mice, normalized for dry lung weight (left). Mean + SEM of n = 4–6 mice per group. Macroscopic images show LPS-induced Evans Blue leakage in the lungs after 18 h (right). b mRNA levels of macrophage activation markers in lung lysates of WT versus Arg-/- mice exposed to intra-tracheal LPS. c mRNA levels of inflammatory cytokine expression in lung lysates of WT versus Arg-/- mice exposed to intra-tracheal LPS. d Representative Western blot and (e) quantification (n = 4) of P(Y207)CrkL, normalized to ß-tubulin, in lung tissue of WT and Arg-/- mice. f Representative immunohistochemistry images using an antibody against p(Y207)CrkL in paraffin lung slices of 5 septic versus 4 non-septic patients. Brown staining indicates p(Y207)CrkL (predominantly observed in the endothelial layer), blue staining indicates hematoxillin. Bars, 25 µm. g Macroscopic images of Evans Blue leakage in the skin after intra-cutaneous administration of PBS or VEGF in WT versus Arg-/- mice. h Quantification of VEGF-induced Evans Blue accumulation in the skin, normalized to that in the presence of PBS only. Means +SEM of n = 4 mice per group. AU, arbitrary units; NS, not significant; *P < 0.05; **P < 0.01

Fig 5: (A) Crkl and pCrkl expression in imatinib treated primary CML lin(-) cells. (B) Real time PCR quantification (fold change) of validated miRNAs in imatinib treated primary CML lin(-) cells with respect to the DMSO control. Expression pattern (fold change) of network enriched miRNAs in CML lin(-) cells treated with imatinib, JAK inhibitor I and combination of imatinib and JAK inhibitor I along with DMSO control. (C) CML patient 1 (D) CML patient 2.