Fig. 3.

The L428A/L429A mutation increases the sensitivity of LCA to intracellular degradation. Non-differentiated SiMa cells expressing the wild-type (WT) LCA, or L428A/L429A mutant, or wild-type LCE were exposed to lactacystin (proteasome inhibitor), bafilomycin (inhibitor of lysosomal V-type ATPase) or MG-132 (inhibitor of proteasomal, lysosomal, and calpain proteases), and the effects of inhibitors on the light chains (LCs) were measured. (A) Cells were incubated with or without lactacystin for 16 h and lysed. GFP-tagged LCs were immunoprecipitated with GFP antibody. Polyubiquitylated LCs were detected with an ubiquitin-specific antibody. The membrane was stripped and re-probed with GFP monoclonal antibody to detect total amounts of the various LCs. Densitometric quantification of the results shows lactacystin-induced accumulation of the polyubiquitylated forms of LCE and the L428A/L429A mutant, but no effect on the wild-type LCA polyubiquitylated forms. (B) Effect of the inhibitors of degradation on the total amount of LCs. Cells expressing the respective GFP-tagged LCs were incubated with cycloheximide in the absence or in the presence of degradation inhibitors for 8 h. The amount of LCs and β-actin in total cell lysates was determined by immunoblotting. Quantification of the results indicates that both MG-132 and bafilomycin protect the L428A/L429A mutant and LCE, but not the wild-type LCA. (C) Downregulation of septin-2 by siRNA increases the rate of LCA degradation. Non-differentiated cells expressing GFP–LCA were treated with septin-2 or control siRNA as described in the Materials and Methods and were incubated with cycloheximide in the absence or presence of MG-132. The amount of LCA, septin-2 and β-actin in total cell lysates was determined by immunoblotting. Quantification of the results indicates that downregulation of septin-2 decreases the amount of mature GFP–LCA in the absence, but not in the presence, of the degradation inhibitor MG-132. Results are mean±s.d. (n = 3). *P<0.01 compared with control (no inhibitors), Student's t-test. WB, western blot analysis; IP, immunoprecipitation; NT, non-transfected cells; ?, unidentified bands; IgG HCh, the heavy chain of the antibody used for immunoprecipitation.