Rpgrip1l deficiency leads to elongation of limb cilia and an increase of the Gli3-190/Gli3-83 ratio but does not alter overall cellular proteasomal activity. (A and B) Western blot analysis of MEFs and E12.5 limbs (WT: n = 3 embryos; Rpgrip1l^−/−: n = 3 embryos, respectively). Gapdh (A) and Actin (B) serve as loading controls. Evaluation of the Gli3-190/Gli3-83 ratio depicts a significant increase in the Rpgrip1l^−/− situation of 3.6-fold (MEFs, A) and 8.1-fold (limbs, B). (C) Proteasomal activity was measured in total cell lysates of MEFs isolated from E12.5 WT (n = 8) and Rpgrip1l^−/− embryos (n = 4). No alteration is detectable in Rpgrip1l^−/− MEFs in comparison to WT MEFs either without ATP (activity of the 20S proteasomal subunit) or after ATP addition (activity of the 20S proteasomal subunit and 26S proteasome together). There is also no alteration in the activity of the 26S proteasome alone (ATP-w/o ATP), demonstrating that the activity of the 19S proteasomal subunit is unchanged in the absence of Rpgrip1l. Error bars show standard error of the mean. *, P < 0.05; **, P < 0.01.