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2012). To analyze the expression pattern of PlAlix during the early stages of development, equal amounts of protein extract from unfertilized eggs and embryos at different times following fertilization (1, 5, 15, 30, 60 and 120?min) were immunoblotted using monoclonal anti-Alix; immuno-positive Alix bands were quantified and normalized against the Ponceau S loading control. As shown Quinapyramine in a representative gel and in the quantitative analysis of PlAlix bands (Fig.?2C, D), a significant increase of PlAlix protein was observed from unfertilized eggs until 60�C120?min after fertilization, indicating a regulated expression during these early stages of development. In these experiments, PlAlix bands are associated with lower molecular mass bands that may be cleavage products of PlAlix (Fig.?2C, gray arrowhead), in line with what has been reported for HuAlix (Pan et?al. 2008). Furthermore, immunoblots of protein extract from embryos at later developmental stages (128-cell, blastula, and gastrula stage embryos) showed that PlAlix is present and its level remains unaltered during these later stages (Fig.?2E). To see if PlAlix expression was similarly regulated Capmatinib chemical structure at the transcriptional level, we analyzed the relative abundance of the PlAlix transcript in unfertilized eggs (UE) and embryos up to 120��. Total RNA from each stage was retro-transcribed and used in qPCR for PlAlix transcripts, normalized with cytochrome oxidase (Cavalieri et?al. 2011). Our results show that, in contrast to protein expression, PlAlix mRNA level decreased to see more the cytosol and localized in enlarged puncta near the nuclei, along the spindle microtubules (Fig.?3B, C, K, L, and M). Thus, PlAlix is localized in cellular structures, likely intracellular vesicles, of different sizes: the smallest are also present in unfertilized eggs, while others of larger size appear soon after fertilization and are more evident after the first division. Close inspection of several specimens pointed out that a number of PlAlix-positive structures are localized close to the cortex or extended from the membrane surface (see arrowheads in Fig.?3E�CG); the number of these sub-cellular compartments increased after fertilization (Fig.?3F, G versus E panel).