Fig 1: Effects of PKD inhibition on cortical actin assembly and p-cofilin expression during meiosis. (A) Representative images of actin distribution in the control and CID755673 treatment groups. White, actin, bar = 100 µm. (B) Fluorescence intensity profiling of actin in the left graphs. Lines in the same direction were drawn through the oocytes, and actin intensities were quantified along these lines. The black arrow in right graphs indicated actin intensity in membrane of oocyte. ZEN Blue Lite software was chosen to perform the analysis. Red, actin, bar = 30 µm. (C) Quantification immunofluorescence intensity levels of actin at the cortex and in the cytoplasm in the control and CID755673 treatment oocytes. Fluorescence intensities were analyzed using ImageJ software. (D) Protein levels of p-cofilin in control and CID755673 treatment oocytes were determined by western blotting. Data are presented as mean ± s.d. from at least three independent experiments. **, significant difference (P < 0.01). ***, significant difference (P < 0.001).
Fig 2: PKD1 expression decreases in aged oocytes. (A) Protein levels of PKD1 in control and postovulatory aging porcine oocytes were determined by western blotting. Rabbit polyclonal anti-PKD1 antibody was adopted. (B) Quantitative analysis of PKD1 expression in control and aging groups. Data are presented as mean ± s.d. from at least three independent experiments. **, significant difference (P < 0.01).
Fig 3: Effects of PKD inhibition on porcine oocyte maturation. (A) The expression of PKD in porcine oocytes. 1, 2, 3 indicates the samples of three replicates. Rabbit monoclonal anti-PKD antibody was adopted. (B) Representative images of cumulus expansion in the control and CID755673-treated groups. Black Crop indicated the status of cumulus expansion in porcine oocytes. Bar = 100 μm (C) Rate of oocytes which extruded the PBI in the control and CID755673 treatment groups, respectively. The rate of PBI extrusion was significantly decreased in 10 μM, 20 μM and 50 μM CID755673-exposed groups. Bar = 30 μm. (D) The proportions of cell cycle distribution were recorded in the control and CID755673 treatment groups. Data were expressed as mean percentage ± s.d. from at least three independent experiments. **, significant difference (P < 0.01), ***, significant difference (P < 0.001).
Fig 4: Effects of PKD inhibition on meiotic spindle configurations and chromosome alignment. (A) Representative images of spindle (green) morphologies and chromosome (blue) alignment in the oocytes from control and CID755673 treatment groups. It showed a typical barrel-shaped spindles and well aligned chromosomes in control oocytes; while, unfocused or tripolar poles spindles with agglutinative/scattered chromosomes were successively showed in PKD inhibition groups. Green,a-tubulin; blue, DNA; Bar = 5 µm. (B) The percentage of spindle/chromosome defects were recorded in control and CID755673-treated oocytes. (C) Protein levels of p-MAPK in control and CID755673 treatment oocytes were determined by western blotting. Data are presented as mean ± s.d. from at least three independent experiments. **, significant difference (P < 0.01). ***, significant difference (P < 0.001).
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