Fig. 2 | Scientific Reports

Fig. 2

From: Loss of Kat2b impairs intraflagellar transport and the Hedgehog signaling pathway in primary cilia

Fig. 2

Kat2b interacts with and regulates the acetylation level of α-tubulin. (a) Western blots showing Kat2b and acetylated α-tubulin expression at 0, 6, 12, and 24 h after serum withdrawal. The graphs showed relative western blotting band intensity of Kat2b and acetylated-α-tubulin normalized each loading controls. Data were collected from three independent experiments. Statistical analysis for normalizing band was conducted by paired t-test. (b) Acetylated α-tubulin expression was decreased in Kat2b silenced NIH/3T3 cells at 6 h and 24 h after serum starvation. The graphs next to immunoblot analysis showed quantified acetylated α-tubulin band intensity. (c) Western blot data showed that overexpression of Kat2b restored the decreased acetylation level of α-tubulin in Kat2b silenced cells at 6 h and 24 h after serum starvation. The relative band intensity of α-tubulin acetylation was displayed through the graphs below. EV indicates empty vector (pCMV-tag2B) and FL-Kat2b indicates full length Kat2b-pCMV-tag2B. (d) Immunoprecipitation data revealed that endogenous Kat2b interacts with α-tubulin in NIH/3T3 cells. (e, f) Fluorescence images showed comparison between ciliary markers under Kat2b silencing with serum starvation 6 h, 24 h. Arl13b marked ciliary membrane (green), ac-α-tubulin, marked ciliary axoneme (red), and nuclei were stained with DAPI (blue). Graphs on right side indicated fluorescence intensity of Arl13b normalized ac-α-tubulin, ac-α-tubulin and Arl13b, respectively. This experiments were independently repeated in triplicate and graphs were analyzed by two-tailed Student’s t-test and values are presented as the mean ± SD. (g) Fluorescence images represented ciliated cells with or without 500 nM garcinol treatment for 6 h with serum starvation. Acetylated α-tubulin and γ-tubulin were stained for primary cilia (green). Nuclei were stained with DAPI (blue). Primary cilia are indicated by white arrows. White arrows indicate primary cilia. Scale bar: 10 μm. The graph showed quantification of the percentage of primary cilia in garcinol-treated cells compared to DMSO-treated NIH/3T3 cells. Counted cell numbers were as follows: DMSO-treated cells n = 287, garcinol-treated cells n = 248. (h) Western blot data showed that the acetylation level of α-tubulin decreased in garcinol-treated NIH/3T3 cells. Graphs on right side showed relative western blotting band intensity of acetylated-α-tubulin normalized α-tubulin and acetylated-α-tubulin normalized Kat2b. (i) Images represented localization of Kat2b within cilia when treated garcinol, and graph displayed mean of Kat2b fluorescence intensity within acetylated-α-tubulin. Data collected from three experiments were quantified graphically.

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