Extended Data Fig. 6: Validation of the antibodies and the iterative indirect immunofluorescence imaging (4i).

Related to Fig. 3. a, b, Single-cell correlation of anti-HA intensity and anti-phospho-Rb (p-T373 for a, p-S608 for b) intensity in cells expressing the Dox-inducible HA-tagged Rb constructs (measured by immunofluorescence). Cells were treated with si-RB1 one day before release to knockdown endogenous Rb, treated with Dox 5 h before release to induce Rb constructs, and fixed 36 h after release with growth media. Cells with the efficient RB1 knockdown were gated based on the correlation between anti-HA and anti-total-Rb. In a, Rb-WT-T373A and ΔCDK-T373 were compared to Rb-WT and ΔCDK, respectively and red reference lines are y = 0.8x + 200. In b, Rb-WT-T373A-S608A and ΔCDK-T373-S608 were compared to Rb-WT and ΔCDK, respectively and red reference lines are y = 0.15x + 200 (n = 3803, 3463, 2857, 3607 cells for Rb-WT, Rb-WT-T373A, ΔCDK-T373, ΔCDK, respectively (a). n = 3863, 2687, 3518, 3499 cells for Rb-WT, Rb-WT-T373A-S608A, ΔCDK-T373-S608, ΔCDK, respectively (b)). c, Workflow of the iterative indirect immunofluorescence imaging (4i) protocol. Representative images and histograms of Rb phosphorylation at T373 and S807/S811. Cells were fixed 14 h after release with starvation media + EGF (20 ng/mL). To validate the signal in the second round of 4i is due to anti-phospho-Rb (p-S807/S811) antibody, 4i was performed with (top) or without (bottom) phospho-Rb (p-S807/S811) staining, while keeping the rest same. The same cells across two rounds of 4i are shown for each protocol (top or bottom). Scale bar = 20 μm (Histograms: n = 2960, 2765 cells for with or without phospho-Rb (p-S807/S811) staining, respectively). d, Phosphorylation Site Preference (PSP) plots showing single-cell correlation of Rb phosphorylation at T373 plotted against S807/S811 or vice versa. Cells were fixed 16 h after release with starvation media + EGF (20 ng/mL) + CDK4/6i (20 nM). Left, cells were stained with anti-phospho-Rb (p-T373) antibody in the first round and anti-phospho-Rb (p-S807/S811) antibody in the second round of 4i. Right, cells were stained with anti-phospho-Rb (p-S807/S811) antibody in the first round and anti-phospho-Rb (p-T373) antibody in the second round of 4i. Each phosphorylation signal was normalized by the total Rb antibody signal in the same cell and each axis was adjusted to the average phosphorylation signal in S phase of 1. Color indicates relative cell population density. A red line shows fitting with a preferential relative phosphorylation or dephosphorylation rate between the two sites (PSP_coeff) (see more details in Methods) (n = 2734, 1727 cells for left and right, respectively. 1 of n = 2 biological replicates).