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. 2015 Mar 23;10(3):e0119423. doi: 10.1371/journal.pone.0119423

Fig 6. Effect of Hook3 siRNA on cytoplasmic transport complexes.

Fig 6

N2A cells were transfected with control siRNA or one out of two Hook3 siRNAs and incubated for 72 h. (a) Knockdown of Hook protein levels. Cells were harvested 72 h after transfection and analyzed by SDS-PAGE and Western blotting. (b) Densitometric analysis of protein immunoreactivity. The averaged Hook3 immunoreactivity of Hook3 siRNA transfected cells is 24% and 15% of control level respectively. C: control RNA, 1: Hook3 siRNA1, 2: Hook3 siRNA2. C. Sedimentation analysis. Lysates of N2A cells transfected with control or Hook3 siRNA2 and sedimentation standards thyroglobulin (19S), katalase (13S) and BSA (3S) were subjected to 5–20% sedimentation analysis followed by SDS-PAGE and Western blotting. Knockdown of Hook3 shifts the presence of p150 to a protein complex smaller than dynactin, whereas between control and Hook3 siRNA treated cells no differences in DIC, p50 and KLC distribution were detected. Sucrose gradient and peak position of sedimentation standards are indicated at the top. (d) Western blot analysis of cell lysates transfected with control or Hook3 siRNA. Antibodies to DIC, KLC, p50, p150 and β-actin indicate no alteration in the total amount of these proteins by treatment with Hook3 siRNA2. A similar result was obtained with Hook3 siRNA1.