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Figure 12


Fig. 12. Effect of Ika on the distribution of CHC. (A) The specificity of the 4A8 monoclonal antibody (Mab) was tested by SDS-PAGE and western blot of pollen tube crude extracts (CE). 4A8 antibody specifically recognized a 167 kDa polypeptide (lane 4A8, arrow). In the control (lane C) assay, the primary antibody was omitted. St, molecular-mass markers. (B) Immunostaining to reveal the effect of Ika on CHC distribution with 4A8 Mab. (Ba-Bb') Medial sections of tubes (1 µm) treated with 3 µM Ika for 15 or 45 minutes showed increased association of CHC with the PM in the apical and subapical regions. (Bc,Bc') In control (Co) experiments, 4A8 antibody gave punctate staining on the PM (arrowheads) and in the cytoplasm. Bars, 20 µm. (C) Cytoplasmic dissection with 3 µM Ika suggested that CHC was found in the microsomal fraction (P2) in preference to the soluble fraction (S2) (row 4A8). In the control microsome preparation, CHC appeared to be more equally distributed between P2 and S2 (row 4A8). Staining of membrane used for CHC detection with an anti-{alpha} tubulin antibody is also reported. (D) Densitometric analysis of anti-CHC western blot with Ika showed an integrated optical density (IOD) of CHC in P2 samples that was almost sixfold greater than that in S2, whereas, in the control experiments, it was only 1.6-fold greater than S2. Error bars indicate standard errors (n=3). 15 µg of proteins were loaded in each lane.





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