Figure 2.
The dynactin complex, but not dynein, is disrupted in the arp1 dsRNA-treated cells. (A) HSS and HSP were prepared from arp1 dsRNA and GFP dsRNA-treated cells. Western blot analysis shows that loss of arp1 leads to reduction in the levels of both p150Glued and p50, whereas KHC and DHC remain unchanged. Note that in the HSS sample, tubulin and actin shown as loading controls display a lower level in the GFP control compared with the Arp1-treated sample, which explains the lower levels of DHC and KHC in that sample. Although the knockdown of Arp1 protein is evident in the HSP where no actin is detected, in the HSS, the Arp1 antibody cross-reacts with actin (see Materials and Methods). (B) Quantitation of transcript amounts was done with RT-qPCR. Arbitrary values of transcripts levels were obtained from duplicate data points and changes in transcript levels for the arp1 RNAi-treated samples were compared with the GFP RNAi-treated samples and normalized to GAPDH. Knockdown of arp1 transcript is shown as percentage (mean of 3 experiments ± SEM). (C–F) HSS and HSP from arp1 dsRNA- and GFP dsRNA-treated cells were sedimented on a 5–20% sucrose gradients. In GFP dsRNA-treated cells, sedimentation of the dynactin subunits Arp1, p150Glued, and p50 are found in a broad peak at ∼17, whereas in the arp1 dsRNA-treated cells, Arp1 levels are reduced and both p150Glued and p50 are found at ∼8S. Note that in the arp1 dsRNA-treated samples, the levels of p50 and p150Glued are reduced, and Western blots for p50 and p150Glued required longer exposure times in D and F (see brackets and A). The Arp1 band detected at ∼8S in both GFP and arp1 dsRNA HSS corresponds to the actin cross-reactivity. This band is not observed in the HSP fraction where no actin can be detected. Dynein and kinesin sedimentation properties are not affected by reduction of Arp1, peaking at ∼17S and ∼8S, respectively. In C and D, arrowhead points to the 400-kDa dynein heavy chain.