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. 2014 May 28;6(4):312–323. doi: 10.1093/jmcb/mju026

Figure 4.

Figure 4

Depletion of Hub1 causes nuclear speckle abnormalities. (A) Co-localization studies in U2OS cells stably expressing GFP-Hub1 (green). Cells were pre-extracted, fixed, and immunostained for splicing proteins (red) using antibodies against nuclear speckle marker phospho-SC35, U1 snRNP (anti-U1A antibody), and snRNP associated Sm proteins (anti-Y12), respectively. Scale bar, 10 µm. (B) Visualization of poly-adenylated mRNA by FISH with fluorescently labeled poly-(dT)-TRITC probes co-stained for nuclear speckles with anti-SC35 antibodies in U2OS cells treated with Hub1 or control RNAi. Scale bar, 10 µm. The nuclear accumulation of poly-adenylated mRNA upon Hub1 knockdown is quantified by measuring the integrated FISH signal as arbitrary intensity units per area in nuclear and cytoplasmic compartments. The box-and-whisker plots represent the quantification of two independent experiments with significant differences between Hub1 and control RNAi treated cells (P as the probability of a two-tailed paired t-test, n> 140 for control cells and n > 239 for Hub1 knockdown cells).