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. 2015 Apr 24;10(4):e0120497. doi: 10.1371/journal.pone.0120497

Fig 4. Inhibition of endogenous PLC affected the clustering of GM1a and phosphorylation of TrkA.

Fig 4

(A) A549 cells were preincubated with 40 μM U73122 (endogenous PLC inhibitor) or U73343 (U73122 analogue) at 37°C for 60 min. The treated cells were stained with BODIPY-GM1a and incubated with 1.0 μg/mL wild-type or H148G alpha-toxin at 37°C for 60 min. The cells were fixed in 4% paraformaldehyde and stained with Hoechst 33342. GM1a (green) and nuclei (blue) were visualized by fluorescence microscopy. Scale bar, 10 μm. (B) A549 cells were preincubated with 40 μM U73122 or U73343 at 37°C for 60 min. The treated cells were incubated with 1.0 μg/mL wild-type or H148G alpha-toxin at 37°C for 60 min. The cells were fixed, permeabilized, and stained with phospho-TrkA antibody and Hoechst 33342. Phospho-TrkA (red) images and nuclei (blue) were visualized by fluorescence microscopy. Scale bar, 10 μm. (C) Bodipy-GM1a fluorescence intensity was measured as described in Materials and Methods. Values represent the mean ± SE; n = 3; *, p < 0.01. (D) Phospho-TrkA fluorescence intensity was measured as described in Materials and Methods. Values represent the mean ± SE; n = 5; *, p < 0.01.