Expressions of Rho A GTPases (Rho A, Cdc42, and Rac1) are altered in lenses of N101D mutant compared with WT. (A) Immunostaining of Rho A in 1- and 5-month-old N101D lenses (b, d), was decreased relative to 1- and 5-month-old WT lenses, (a, c). In contrast, the Cdc42 immunostaining in 1-month-old N10D lenses (f) was increased compared with 1-month-old WT lenses (e). Slight increase in Cdc42 immunostaining in 5-month N101D lenses (h) compared to 5-month WT lenses (g). Similarly, Rac1 immunostaining in, 1- and 5-month-old N101D lenses (j, l) increased relative to 1-and 5-month-old WT lenses (i, k). Nuclei were counter-stained with DAPI. Scale bar: 60 μm. (B) Western blot analysis was used to determine levels of Rho A, Cdc42, and Rac1 in WT and N101D lenses. The 1- and 5-month-old WT and N101D lenses were homogenized in RIPA buffer as described in Materials and Methods and probed with anti-Rho A, Cdc42, and Rac1 antibodies. Equal amounts of protein loading was confirmed by probing for anti–β-actin antibody. Four lenses from two animals for each age were used in the experiment.