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. 2011 Sep 12;121(10):3965–3980. doi: 10.1172/JCI58552

Figure 2. Prolonged loss of CD2AP leads to expression of cytosolic CatL.

Figure 2

(A) Actin cytoskeleton and FA organization in fully differentiated low- and high-Tgfb1 Cd2ap–/– cells. Note the loss of well-defined stress fibers and dramatic increase in number of transverse arcs in high-Tgfb1 Cd2ap–/– cells. FAs and F-actin were visualized with anti-paxillin antibodies and rhodamine-phalloidin, respectively. (B) Low-Tgfb1 Cd2ap–/– podocytes could be transformed into high-Tgfb1 Cd2ap–/– podocytes by high TGF-β1 levels. WT and low-Tgfb1 Cd2ap–/– passage cells were treated with 5 ng/ml TGF-β1 in the media for 24 hours. Actin cytoskeleton was monitored by staining cells with rhodamine-phalloidin, and dendrin localization was monitored using anti-dendrin antibody (green). (C–E) mRNA levels for Tgfb1 (C) and Ctsl (D and E), determined by RT-PCR in podocytes. When indicated, cells were treated with 5 ng/ml TGF-β1 for 24 hours or with 50 μM LPS for 24 hours. Podocytes expressing Actn4 mutant are also shown in E. (F) Subcellular fractionation of low- and high-Tgfb1 Cd2ap–/– podocytes in isotonic sucrose. Total proteins from soluble (S) and particulate (P) fractions were probed for CatL and for the lysosomal protein markers Lamp-2 and mannosidase (Manno), then analyzed by Western blotting. GAPDH was used as a loading control. Strong cytosolic CatL induction (asterisk) was observed in both soluble and pellet fractions of high-Tgfb1 Cd2ap–/– podocytes. Scale bars: 20 μm.