Figure 1. Nodes are stable scaffolds... | Rockefeller University Press

Figure 1.

$Figure 1. Nodes are stable scaffolds that facilitate inhibition of Wee1. (A) Wee1 phosphorylation is disrupted in cdr1Δ and cdr2Δ cells. Whole-cell extracts were separated by SDS-PAGE, and endogenous Wee1 was detected by Western blotting. (B) Localization of Cdr2-mEGFP and Cdr2ΔC-mEGFP in cells. Maximum-intensity projections from z series are shown. (C) Wee1 phosphorylation is disrupted in cdr2ΔC cells. Whole-cell extracts were analyzed as in A. Data in A and C are taken from the same Western blot, and the wee1Δ lane is the same in both. (D) Visualization of node-like puncta by TIRF microscopy in whole-cell extracts from the indicated strains. Bars, 5 µm. (E) Detergent extracts from cdr2-FLAG or cdr2ΔC-FLAG cells were subjected to velocity sucrose gradient sedimentation, and fractions were probed against the FLAG tag or against Cdr2(pT166). Fraction 1 corresponds with the top of the gradient and contains smaller complexes; fraction 12 corresponds with bottom of the gradient. S values were determined using size standards run on identical gradients. (F) Quantification of the number of Cdr2 and Cdr1 molecules per node (means ± SD; n > 65 each) based on superresolution live-cell fluorescence microscopy.$

Nodes are stable scaffolds that facilitate inhibition of Wee1. (A) Wee1 phosphorylation is disrupted in cdr1Δ and cdr2Δ cells. Whole-cell extracts were separated by SDS-PAGE, and endogenous Wee1 was detected by Western blotting. (B) Localization of Cdr2-mEGFP and Cdr2ΔC-mEGFP in cells. Maximum-intensity projections from z series are shown. (C) Wee1 phosphorylation is disrupted in cdr2ΔC cells. Whole-cell extracts were analyzed as in A. Data in A and C are taken from the same Western blot, and the wee1Δ lane is the same in both. (D) Visualization of node-like puncta by TIRF microscopy in whole-cell extracts from the indicated strains. Bars, 5 µm. (E) Detergent extracts from cdr2-FLAG or cdr2ΔC-FLAG cells were subjected to velocity sucrose gradient sedimentation, and fractions were probed against the FLAG tag or against Cdr2(pT166). Fraction 1 corresponds with the top of the gradient and contains smaller complexes; fraction 12 corresponds with bottom of the gradient. S values were determined using size standards run on identical gradients. (F) Quantification of the number of Cdr2 and Cdr1 molecules per node (means ± SD; n > 65 each) based on superresolution live-cell fluorescence microscopy.