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. 2016 Oct 4;17(11):1624–1640. doi: 10.15252/embr.201642378

Figure EV5. Heat shock responses in w −, dCYLD mutant, and LUBEL knockdown (KD) flies.

Figure EV5

  1. Immunoblotting for total Ub chains in heat‐shocked w − flies in Fig 6A using anti‐pan Ub antibody.
  2. mRNA expression of poly‐ubiquitin gene ubi‐p63E in LUBEL mutant flies, untreated, or heat shocked. 10 male and 10 female adult flies were heat shocked for 1 h and RNA was isolated and Ubi‐p63E mRNA level was measured by qPCR. Rp49 was used as reference and untreated samples were used as calibrator for each fly line to calculate the expression ratio. Data are analyzed by two‐way ANOVA with multiple comparison, and presented as mean ± SD (****P < 0.001). Representative of three experiments is shown.
  3. Expression of LUBEL mRNA in heterozygous flies (Tub‐Gal4/+ and Mef2‐Gal4/+) and UAS‐shLUBEL (Tub‐Gal4 > LUBEL and Mef2‐Gal4 > LUBEL) flies detected by qPCR. The primers that target N‐terminal region (left panel) or catalytic region (right panel) of LUBEL were used. Rp49 was used as reference, and GD control fly line was used as calibrator to calculate the expression ratio. Data were analyzed using unpaired t‐test; values represent mean ± SD (**P < 0.01, ***P < 0.001), (n = 3). Representative of three independent experiments are shown.
  4. Survival of dCYLD mutant flies upon heat shock. Survival curve of heat‐treated w − and dCYLD mutant flies is shown. 15 male and 15 female files per each fly line were used in this assay. Curve comparison tests indicated the difference is not significant. Representative data are shown from four independent experiments.
  5. Heat‐induced mRNA expression of HSP70. w −, CC/SS #2, and delR2 flies were heat treated for 30 min and mRNA HSP70 was quantified by qPCR. Rp49 was used as a reference and w − untreated sample was used as calibrator to calculate the expression ratio. Data are analyzed by two‐way ANOVA with multiple comparison, and presented as mean ± SD (****P < 0.0001). Representative of three experiments is shown.
  6. Repeat of muscle‐specific LUBEL KD using B24‐Gal4 driver. A heat‐hock survival assay was performed as Fig 6C, using 24B‐Gal4 driver line to knockdown LUBEL in the muscle. P‐values calculated by Gehan–Breslow–Wilcoxon test: Tub‐Gal4 < 0.0001 (****), 24B‐Gal4 = 0.0066 (**). knockdown efficiency for the catalytic region, analyzed as in (C), is shown on the right graph. Representative of three experiments is shown.