Intraflagellar Transport (IFT) Protein IFT25 Is a Phosphoprotein Component of IFT Complex B and Physically Interacts with IFT27 in Chlamydomonas (original) (raw)
Figure 6
The majority of IFT25 and IFT27 are not integrated into the IFT complex B in the cell body.
(A). Wild-type flagella extracts or cell body lysates of the bld2 mutant were fractionated on a 10–25% sucrose gradient. The fractions were then separated by SDS-PAGE. Immunoblots probed with antibodies against several IFT particle proteins as indicated to the left of the blots. Flagellar IFT25 and IFT27 were found in two peaks, a major peak at 16 S fractions, and a minor peak at much lighter fractions. IFT46, a complex B subunit, only peaked at 16S fractions. Only a portion of whole-cell IFT25 and IFT27 was integrated into the preassembled IFT complex B in the cell body. IFT particle proteins, including complex A subunit IFT139 and complex B subunits IFT172, IFT81, and IFT74.were found entirely in 16S fractions. In contrast, IFT25 and IFT27 had two peaks on the gradient. The Western blots shown for IFT25 were exposed for 1 minutes or 5 minutes to ensure that phosphorylated IFT25 was detected in the 16S fractions. (B). Ponceau S staining of the membrane showing the protein profiles of whole cells, cell bodies, and flagella. Flagellar proteins were isolated from 5×106 (5×), 1.5×107 (15×), and 5×107 (50×) cells. Whole cell or cell body proteins were isolated from 1×106 (1×), 3×105 (0.3×), and 1×105 (0.1×) cells. (C). The distributions of IFT25 and IFT27 proteins among whole cells, cell bodies, and flagella. The same or a duplicated membrane shown in B was probed with antibodies as indicated to the left. (D). The intensity of each lane or band stained by Ponceau S or antibodies. Photoshop CS3 software was used for the measurement.