2D-DIGE proteomic analysis of flagellar protein accumulation and depletion in dhc1b-3 cells at 21 and 34°C. (A) Fluorescence image of the 2D-DIGE analytical gel (dhc1b-3 21C = green; dhc1b-3 34C 18 h = red; for overlays of all three samples see Fig. S5). The large yellow spot at ∼50 kD is tubulin. (B and C) Depletion and accumulation profiles for every spot that was identified by mass spectrometry. Fold change is a spot’s relative fluorescence between two samples on the analytical gel (1 = equal abundance for both samples). Class D1 = depleted in only dhc1b-3 21C vs. wild-type 21C (accumulated in dhc1b-3 34C 18 h vs. dhc1b-3 21C); class D2 = depleted in both dhc1b-3 21C vs. wild-type 21C and dhc1b-3 34C 18 h vs. wild-type 21C; class D3 = depleted in only dhc1b-3 34C 18 h vs. wild-type 21C (depleted in dhc1b-3 34C 18 h vs. dhc1b-3 21C); class A1 = accumulated in only dhc1b-3 21C vs. wild-type 21C (depleted in dhc1b-3 34C 18 h vs. dhc1b-3 21C); class A2 = accumulated in both dhc1b-3 21C vs. wild-type 21C and dhc1b-3 34C 18 h vs. wild-type 21C; class A3 = accumulated in only dhc1b-3 34C 18 h vs. wild-type 21C (accumulated in dhc1b-3 34C 18 h vs. dhc1b-3 21C). (D) The profiles of proteins identified in multiple spots that group into more than one class, suggesting IFT-related protein modifications. Negative pI shifts (minus sign omitted) and MW shifts, both relative to the spot with the highest pI, are noted.