FCS can be used to measure cytoplasmic protein concentrations in the early Drosophila embryo. (A i) Graph shows the FCS-measured concentration (mean ± SEM) of Sas-6-GFP expressed transgenically from its endogenous promoter in embryos laid by females expressing either: 1 copy of the transgene (1X); 2 copies of the transgene (2X—shown for two different transgenic lines); four copies of the transgene (4X). Each data point represents the average of 4–6× 10-s recordings from an individual embryo (N ≥ 14). (ii) Western blots of 0–2 h old embryos laid by the females described in A (i). (B i) Graph shows the FCS-measured concentration (mean ± SEM) of Ana2-mNG expressed from a CRISPR/Cas9 knock-in line as either a heterozygote (1X) or homozygote (2X). Each data point represents the average of 4–6× 10-s recordings from an individual embryo (N ≥ 18). (ii) Western blots of 0–2 h old embryos laid by the females described in B (i). (C i) Graph shows FCS-measured cytoplasmic concentrations (mean ± SEM) of WT Ana2-mNG, eAna2(∆CC)-mNG and eAna2(∆STAN)-mNG (all in an ana2+/− heterozygous background). Each data point represents the average of 4–6× 10-s recordings from an individual embryo (N = 14). (ii) Western blots of 0–2 h embryos showing the expression levels of endogenous Ana2, a homozygous WT Ana2-mNG knock-in line, and transgenic lines expressing either WT Ana2-mNG, eAna2(∆CC)-mNG and eAna2(∆STAN)-mNG (all in an ana2+/− heterozygous background). (D i) Graph compares FCS-measured cytoplasmic Ana2-mNG concentrations (mean ± SEM) in the transgenic WT eAna2-mNG (generated by P-element mediate transformation and expressed in an ana2-/- mutant background) and CRISPR/Cas9 knock-in Ana2-mNG lines. Each data point represents the average of 4–6× 10-s recordings from an individual embryo (N ≥ 11). (ii) Western blots of 0–2 h embryos comparing the expression levels of Ana2-mNG in the eAna2-mNG transgenic line and the Ana2-mNG knock-in line generated by CRISPR/Cas9. For Western blotting, actin or Cnn are shown as loading controls. Prominent non-specific bands are highlighted (*). A representative blot is shown from at least two technical repeats. Statistical significance was assessed using an unpaired t test with Welch’s correction (for Gaussian-distributed data) or a Mann-Whitney test (****, P < 0.0001; **, P < 0.01).
Figure S1.

FCS can be used to measure cytoplasmic protein concentrations in the early Drosophila embryo. (A i) Graph shows the FCS-measured concentration (mean ± SEM) of Sas-6-GFP expressed transgenically from its endogenous promoter in embryos laid by females expressing either: 1 copy of the transgene (1X); 2 copies of the transgene (2X—shown for two different transgenic lines); four copies of the transgene (4X). Each data point represents the average of 4–6× 10-s recordings from an individual embryo (N ≥ 14). (ii) Western blots of 0–2 h old embryos laid by the females described in A (i). (B i) Graph shows the FCS-measured concentration (mean ± SEM) of Ana2-mNG expressed from a CRISPR/Cas9 knock-in line as either a heterozygote (1X) or homozygote (2X). Each data point represents the average of 4–6× 10-s recordings from an individual embryo (N ≥ 18). (ii) Western blots of 0–2 h old embryos laid by the females described in B (i). (C i) Graph shows FCS-measured cytoplasmic concentrations (mean ± SEM) of WT Ana2-mNG, eAna2(∆CC)-mNG and eAna2(∆STAN)-mNG (all in an ana2+/− heterozygous background). Each data point represents the average of 4–6× 10-s recordings from an individual embryo (N = 14). (ii) Western blots of 0–2 h embryos showing the expression levels of endogenous Ana2, a homozygous WT Ana2-mNG knock-in line, and transgenic lines expressing either WT Ana2-mNG, eAna2(∆CC)-mNG and eAna2(∆STAN)-mNG (all in an ana2+/− heterozygous background). (D i) Graph compares FCS-measured cytoplasmic Ana2-mNG concentrations (mean ± SEM) in the transgenic WT eAna2-mNG (generated by P-element mediate transformation and expressed in an ana2-/- mutant background) and CRISPR/Cas9 knock-in Ana2-mNG lines. Each data point represents the average of 4–6× 10-s recordings from an individual embryo (N ≥ 11). (ii) Western blots of 0–2 h embryos comparing the expression levels of Ana2-mNG in the eAna2-mNG transgenic line and the Ana2-mNG knock-in line generated by CRISPR/Cas9. For Western blotting, actin or Cnn are shown as loading controls. Prominent non-specific bands are highlighted (*). A representative blot is shown from at least two technical repeats. Statistical significance was assessed using an unpaired t test with Welch’s correction (for Gaussian-distributed data) or a Mann-Whitney test (****, P < 0.0001; **, P < 0.01).

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