Extended Data Fig. 2: Single-cell phosphoprotein analyses of patient-derived B-ALL samples reveal segregation of STAT5 and ERK phosphorylation to competing clones.

a–e, For single-cell phosphoprotein analyses, scWest chips were used to capture individual cells and perform size-based protein separation. Each chip includes 16 arrays of 400-well blocks (6,400 wells) on a polyacrylamide gel. Single-cell suspensions of patient-derived B-ALL cells were loaded onto the scWest chip and inserted into Milo (ProteinSimple) for cell lysis, size-based protein separation and UV capture to immobilize protein bands. a, A representative scanned image of the scWest chip probed with histone H3 antibodies to confirm cell occupancy, followed by fluorescent secondary antibodies (n = 16 independent biological samples). b, c, Chips were probed with anti-STAT5-pY694 and anti-ERK-pT202/Y204 antibodies followed by fluorescent secondary antibodies for simultaneous detection of both phosphoproteins. At the left are representative images of signals observed for STAT5-pY694 and ERK-pT202/Y204. At the right, fluorescence intensity was plotted against distance from the well centre (peak location, in μm). In addition to histone H3, chips were stained for DNA using TOTO-1 dye to verify cell occupancy. Each signal was inspected to confirm that it was associated with a peak located at the correct distance from the well centre (n = 16 independent biological samples, each in triplicate). Chips were scanned using a microarray scanner, and peak identification was performed using Scout Software (ProteinSimple). For single-cell western analyses, optimal cell loading is achieved when 2% or fewer wells contain multiple cells (https://www.proteinsimple.com/milo.html). d, e, Single-cell phosphoprotein analyses for STAT5-pY694 and ERK-pT202/Y204 were performed for patient-derived B-ALL samples (six independent biological samples, each in triplicate) with anti-STAT5-pY694 and anti-ERK-pT202/Y204 antibodies for simultaneous detection of both STAT5 and ERK phosphorylation. scWest chips were then probed for histone H3 and TOTO-1 (DNA stain) to verify cell occupancy. Scout Software (ProteinSimple) was used for peak identification and data analysis. Each data point was inspected to confirm that the signal detected was associated with a peak located at the correct peak location (distance from the well centre). d, Heatmaps illustrating cells that express STAT5-pY694 (green) and/or ERK-pT202/Y204 (red). Sample names and genetic characteristics are shown at the top of each heatmap. e, Tables summarizing the number of cells expressing neither STAT5-pY694 nor ERK-pT202/Y204 and the number of cells expressing either STAT5-pY694 or ERK-pT202/Y204, or in rare cases, both. For single-cell western analyses, optimal cell loading is achieved when 2% or fewer wells contain multiple cells.