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Figure 4


Fig. 4. siRNA knock-down of CLIMP-63 expression led to an increased lateral mobility of the TCs in M3/18 cells. (A) Western blot analysis of M3/18 cells transfected with siRNA#1 (lane 1), siRNA#2 (lane 2), siRNA#3 (lane 3), mock transfected (lane 4) or untreated (lane 5). The samples were collected 4 days after transfection. Staining of the western blot with anti-CLIMP-63 pAb shows that siRNA#1 and siRNA#2 are able to silence the expression of CLIMP-63 in M3/18 cells (lanes 1 and 2), wheras siRNA#3 had no effect on CLIMP-63 expression (lane 3) when compared with mock-transfected (lane 4) or untreated (lane 5) cells. The staining of the same blot with anti-calnexin or anti-ribophorin I pAbs revealed that expression levels of these proteins were not affected by the CLIMP-63 knock-down. (B) Immuno-fluorescent microscopy of mock-transfected cells (a-c) or cells transfected with siRNA#2 (d-f). The cells were collected on day 1,3 and 5 after transfection, fixed in 2% PFA in PBS and stained with anti-CLIMP-63 pAb followed by goat anti-rabbit IgG conjugated to Alexa Fluor-543. The images were collected with the LSM510 microscope equipped with a HeNe1 laser and a Plan-Apochromate 100x/1.4 oil DIC lens at scan-zoom set at `1' and line average set at `4'. The pinhole was set to obtain 0.5 µm thick optical slices. The immunofluorescence microscopy results support western blot data showing that siRNA#2 can effectively knock down of CLIMP-63 expression.Bar, 20 µm. (C) FRAP analysis of untreated M3/18 cells confirmed our previous findings that the lateral mobility of the TCs is very low (Deff=0.063 µm2/second). FRAP analysis of mock-transfected cells performed at the day 4 after transfection suggested that electro-shocking of the cells suspended in transfection buffer had no effect on the TC mobility (Deff=0.067 µm2/second, P(T<=t)=0.427). However, transfection of the cells with siRNA#2 led to an increased diffusion rate of the TCs (Deff=0.137 µm2/second, P(T<= t)<0.001). Knock-down of CLIMP-63 with siRNA had no effect on the lateral mobility of the ER membrane protein LBR-GFP.





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