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. 2015 Dec 7;291(5):2170–2180. doi: 10.1074/jbc.M115.658591

FIGURE 2.

FIGURE 2.

Impaired adhesion and spreading of 4.1G−/− MEF cells on fibronectin. A, cells were plated on fibronectin-coated 96-well plates and incubated for 0.5, 1, and 3 h. The adherent cells were stained with crystal violet, and the staining intensity was quantified by spectrophotometry at 560 nm. The results are mean ± S.E. of three independent experiments. B, cells were plated on fibronectin-coated 4-well chambers and allowed to spread for 3 h. The cells were labeled with Alexa Fluor 488-conjugated wheat germ agglutinin (Invitrogen), and the images were collected using a Zeiss Axiophot wide-field epifluorescence microscope. C, the mean surface area from 35 individual cells was calculated using LSM 5 Pascal software. The data are shown as mean ± S.E. of three experiments. One-tailed Student's t tests were applied to test the statistical significance of the data, with the p value for 4.1G+/+ cells versus 4.1G−/− cells at 1 h being p < 0.0001 and of 4.1G+/+versus 4.1G−/− cells at 3 h being p < 0.0001. D, immunoblot analysis of 4.1G−/− MEF cells transfected with GFP or GFP-4.1G. Total lysates (35 μg of protein) were probed with polyclonal rabbit antibodies against GFP. E, 4.1G−/− MEF cells transfected with GFP or GFP-4.1G were plated on FN-coated coverslips and allowed to spread for 3 h. The cells were labeled with Alexa Fluor 488-conjugated wheat germ agglutinin (Invitrogen), and the images were collected using a Zeiss Axiophot wide-field epifluorescence microscope. Scale bars = 20 μm. F, the mean surface area of 35 individual cells was calculated using LSM 5 Pascal software. The data shown are mean ± S.E. of three experiments. One-tailed Student's t tests were applied to test the statistical significance of the data, with the p value for 4.1G −/− cells transfected with GFP-4.1G versus 4.1G−/− cells being p < 0.0001 and of 4.1G−/− cells transfected with GFP-4.1G versus 4.1G −/− cells transfected with GFP being p < 0.0001. G, analysis of 4.1G and 4.1N upon GFP-4.1G rescue. a, immunoblot. Total lysates were probed with antibodies as indicated. b, quantitative analysis of endogenous 4.1G and GFP-4.1G. GFP-4.1G was calculated on the basis of band intensity and transfection efficiency. c, quantitative analysis of 4.1N. *, p < 0.05.