Figure 4. Reconstitution of low specific activity containing PIP4K: hPIP4K2C restores the cell size reduction seen in dPIP4K²⁹.

(A) PI5P kinase activity assay. (i) Immunoblot from lysates of S2R+ cells expressing hPIP4K. (ii) S2R+ cell-free lysate expressing PIP4 kinases (10 μg) from indicated human isoforms was incubated for 16 h at 30°C with physiological (6 μM) concentration of PI5P and 0.5 μCi [γ³²P] ATP. The assay reaction was terminated by adding 2.4 M HCl followed by methanol and chloroform. The lipids were extracted and resolved on TLC plate using chloroform:methanol:water:25% ammonia (45:35:8:2). UTC, untransfected cells; hPIP4K2A^WT, human PIP4 kinase α isoform wild-type; hPIP4K2C^WT, human PIP4Kinase γ isoform wild-type; hPIP4K2C ^KD. (B) Graph representing average cell size measurement (μm³) as mean ± S.E.M. for heterologous reconstitution of hPIP4K2C as compared with AB1Gal4/+;dPIP4K²⁹, n=7 or more, **P-value <0.05. (C) Immunoblot showing the comparison of expression between of hPIP4K2C^WT or hPIP4K2C^KD from salivary gland lysates and probed with anti-GFP. Tubulin was used as the loading control. (D) Confocal images of salivary glands expressing hPIP4K2C^WT or hPIP4K2C^KD where green represents GFP-tagged transgenic protein expressed in dPIP4K²⁹ and DAPI stains nucleus shown in blue. Scale bar indicated at 20 μm. Images are contrast adjusted for representation. (E) Graph representing average cell size measurement (μm³) as mean ± S.E.M. for heterologous reconstitution of hPIP4K2C^WT or hPIP4K2C ^KD as compared with AB1Gal4/+;dPIP4K²⁹, n=7 or more, *P-value <0.01, **<0.05.