Figure 2

The C19ORF5 protein does not mediate NORE1A binding to microtubules in vitro. A: C19ORF5 protein was depleted by RNA interference in HEK293 cells expressing HA-tagged NORE1A. Equal amounts of cell extracts were probed for C19ORF5 (upper panel) or used to examine the ability of NORE1A to bind to microtubules using the microtubule cosedimentation in vitro assay. B: HEK293 cell extract was immunodepleted of the C19ORF5 protein by incubation with 4G1 antibody followed by Protein A/G plus agarose, or mock-depleted. Equal amounts of C19ORF5-depleted and mock-depleted extracts were probed for C19ORF5 (upper panel). The ability of purified FLAG-NORE1A to interact with microtubules after preincubation with C19ORF5-immunodepleted extract (lanes 1–2), mock-immunodepleted extract (lanes 3–4), or purified FLAG-C19ORF5 (lanes 5–6) was examined as described in the Methods. C. C19ORF5 protein was depleted by RNA interference in A549 cells expressing GFP-tagged NORE1A. Equal amounts of cell extracts were probed for C19ORF5 (upper panel) or used to examine the ability of NORE1A to bind to microtubules as described in Figure 1 (two middle panels). Lower panel, the ability of GFP moiety, expressed alone in A549 cells, to bind to microtubules was examined.