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. 2024 Mar 1;227(1):iyae033. doi: 10.1093/genetics/iyae033

Fig. 4.

Fig. 4.

FBF-1/-2, but not PUF-3, protein levels are reduced in csn(lf) mutants. a) Western blot analysis of 3xv5::fbf-2(q932), referred to throughout the figure as wild type, and respective 3xv5::fbf-2(q932); csn(lf) adult mutant worm lysate reveals reduced levels of FBF-1 (top panels) and 3xV5::FBF-2 (bottom panels). Alpha-tubulin is used for normalization as a loading control. Endogenous FBF-1 and epitope-tagged endogenous 3xV5::FBF-2 are detected by anti-FBF-1 and anti-V5. b, c) Quantitation of total FBF protein level for each 3xv5::fbf-2(q932); csn(lf) mutant. A ratio of FBF/tubulin Western blot intensities in each strain was normalized to the ratio in 3xv5::fbf-2(q932). Differences in protein level were evaluated by 1-way ANOVA with Tukey's posttest. Gray asterisks denote statistical significance compared to wild type, and blue bracket and asterisk denote statistical significance comparing csn-2(lf) to csn-5(lf) (*P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001). Number of biological replicates (N) is indicated at the bottom of the graphs. Mean group values are shown as lines, and error bars denote standard deviation. d–k) Distal germlines of adult wild type (3xv5::fbf-2(q932)) and respective 3xv5::fbf-2(q932); csn(lf) mutants are dissected and stained with anti-FBF-1 (d–g) or anti-V5 (h–k) and DAPI to visualize protein levels of FBF-1/-2. The individual FBF and DAPI channels are also shown separately for better contrast. l, m) Distal germlines of adult 3xv5::puf-3 and csn-5(lf) 3xv5::puf-3 dissected and stained with anti-V5 and DAPI. Germlines are outlined with dashed lines. Scale bars: 10 μm. n) Peak 3xV5::PUF-3 protein levels in the distal germlines were determined by immunofluorescence and scaled to average peak levels in the 3xv5::puf-3 strain. Each data point represents a single germline. Differences in peak 3xV5::PUF-3 levels were evaluated by Student's t-test. Asterisks denote statistical significance (****P < 0.0001). Number of germlines scored (N) is indicated at the bottom of the graphs. PUF-3 abundance appeared higher in csn-5(lf) than in csn-5(+) across the distal 14 cell diameters (E.O., unpublished results). Mean group values are shown as lines, and error bars denote standard deviation. Data reflect 3 biological replicates.