Supplemental Materials

This article contains the following supporting material:

• Figure S1 - Estimation of the relative expression levels of MHC-IIA and MHC-IIB in MRC-5 SV1 TG1 cells. MHC-IIA and MHC-IIB were quantitatively immunoprecipitated from a constant volume of lysate using anti-MHC-IIA pAb and anti-MHC-IIB (C-term) pAb, respectively. The immunoprecipitates were eluted in a constant volume of 2 „e SDS lysis buffer. (A) The immunoprecipitates and their supernatant were analyzed by immunoblotting. The results revealed that MHC-IIA and MHC-IIB were immunoprecipitated specifically and quantitatively. As a result, each isoform was completely immunoprecipitated from the lysates. (B) Various amounts of immunoprecipitates of MHC-IIA relative to that of MHC-IIB were analyzed by immunoblotting. The figure indicates the relative volume of each sample used for the analysis. Anti-pan-myosin II mAb (Covance, Berkeley, CA) and ECL Plus western blotting detection reagents were used for detection. (C) Logarithmic values of the signal intensities for each band were plotted against the relative volume of MHC-IIA. From the standard curve and the signal intensity of MHC-IIB (open circle), we estimated that the relative expression level of MHC-IIA was fifteen times higher than that of MHC-IIB. (D) Schematic explanation of the relative expression levels of the rod fragments and endogenous myosin II molecules. The relative expression level of GFP-ARF296 was approximately three times higher than that of endogenous MHC-IIA as estimated from following calculation; the value 1.8, which is a ratio of signal intensity of GFP-ARF296 (Figure 3H, second panel, lane 1) to that of endogenous MHC-IIA (Figure 3H, forth panel, lane 1), divided by the value 0.6, which is the transfection efficiency of the experiment, equals three. The ratio of signal intensity of GFP-BRF305 (Figure 3H, top panel, lane 4) to that of GFP-ARF296 (Figure 3H, top panel, lane 1) was two. The relative expression level of GFP-ARF296 against the endogenous MHC-IIB was calculated as 45 by multiplying the value 15 (the relative expression level of MHC-IIA against MHC-IIB) and the value three (described above). The relative expression level of GFP-BRF305 against the endogenous MHC-IIB was calculated as 90 by multiplying 45 (described above) and the value two (described above). Because the signal of endogenous MHC-IIB was too weak to quantify (maybe outside the detection limit in densitometric analysis), we could not obtain reliable value from the direct comparison between GFP-BRF305 and endogenous MHC-IIB. The values indicated with solid arrow and dashed arrow were estimated by the direct quantitation using densitometer and by the indirect quantitation from the calculation described above, respectively.
• Figure S2 - The phenotype of MRC-5 SV1 TG1 cells treated with MHC-IIB specific siRNA. MHC-IIB specific siRNA (sequence was described in Bao et al., 2005) was chemically synthesized by Dharmacon, Inc. (Lafayette, CO) and was transfected into the cells by Lipofectamine 2000. (A) After 72 hours, the transfected cells showed aberrant cell shape (white arrowheads). Bar, 20 μm. (B), (C) Immunoblot analysis revealed that the expression level of MHC-IIB decreased to 12.8 ± 3.3% at 72 hours after the transfection. Error bar indicates ± standard deviation of three independent experiments. By using fluorescein-conjugated siRNA (siGLO RISC-Free siRNA: Dharmacon, Inc., Lafayette, CO), we confirmed that more than 95% of the cells were transfected with the siRNA. (D) Percentage of cells with aberrant shapes seen 72 hours after transfection was calculated from at least 100 cells. siGLO RISC-Free siRNA was used as control. Error bars indicate ± standard deviation of three independent experiments. The morphological scoring of cells was performed in a double-blind manner.
• Figure S3 - Phenotype of blebbistatin-treated MRC-5 SV1 TG1 cells. The cells were treated with 0.08% DMSO as control (A) or 20 μM (±)-blebbistatin (Calbiochem, La Jolla, CA) (B) for 60 min. Bar, 20 μm.
• Movie1 - Locomotion of the MRC-5 SV1 TG1 cell expressing GFP-BRF305. GFP image was shown at the beginning of the movie to indicate the GFP-BRF305-expressing cell.
• Movie2 - Cytokinesis of the MRC-5 SV1 TG1 cell expressing GFP-BRF305. GFP image was shown at the beginning of the movie to indicate the GFP-BRF305-expressing cell.
• Movie3 - Cytokinesis of the MRC-5 SV1 TG1 cell expressing GFP as control. GFP image was shown at the beginning of the movie to indicate the GFP-expressing cell.
• Movie4 - Locomotion of the MRC-5 SV1 TG1 cell expressing GFP-ARF296exNC. GFP image was shown at the beginning of the movie to indicate the GFP-ARF296exNC-expressing cell.