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Western blot analysis of αA-crystallin-wt and its mutants, R12C, R21L, R21W, R49C, R54C, R116C and R116H expressed in HeLa cells.

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posted on 20.02.2013, 11:18 by Ilangovan Raju, Edathara C. Abraham

A. Western blot analysis of HeLa cells individually expressed with αA-Crystallin: Cells were individually transfected with YFP-tagged αA-wt and mutants. After 48 h transfection, cells were lysed and 5 µg of total protein was subjected to western blot. The blot was probed with human anti-αA antibody. The same blot was stripped and re-probed for β- actin to serves as a loading control. Nearly a similar level of expression of αA was evident in each of the transfected cells. The * indicates the non-speficific band. B: Western blot analysis of HeLa cells co-expressed with CFP-tagged αA-wt and YFP-tagged αA-wt or its mutants. Cells were co-transfected with YFP-tagged αA-wt and mutants with CFP-tagged αA-wt. After 48 h transfection, cells were lysed and 5 µg of total protein was subjected to western blot. The blot was probed with an antibody against human αA and the same blot was stripped and re-probed for actin. A similar level of expression of αA was detected in each group. C: Western blot analysis of HeLa cells co-expressed with CFP-tagged αB-wt and YFP-tagged αA-wt or its mutants. After 48 h transfection, 5 µg of total protein was subjected to western blot. The blot was probed with an antibody against human αA and the same blot was stripped and re-probed for anti-αB. The same blot was again stripped with anti-β-actin for loading control. The level of both αA and αB were nearly equal in each of the transfected cells.

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