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. 2011 Mar 8;124(7):1032–1042. doi: 10.1242/jcs.075168

Fig. 3.

Fig. 3.

Mutation of KLC1ser460 to alanine or aspartate residues alters the binding of KLC1 to calsyntenin-1. (A) Cells were co-transfected with EGFP–calsyntenin-1 (calsyntenin) and either FLAG–KLC1wt, FLAG–KLC1ser460ala (KLC1ala) or FLAG–KLC1ser460asp (KLC1asp). KLC1 was immunoprecipitated (IP) using the anti-FLAG antibody and calsyntenin-1 was immunoprecipitated using the anti-GFP antibody. The samples were then probed on immunoblots for KLC1, using the anti-FLAG antibody, and for calsyntenin-1, using the anti-GFP antibody. To demonstrate the specificity of the immunoprecipitations, cells were also singly transfected with either FLAG–KLC1wt or EGFP–calsyntenin-1. Samples of the input lysates and immunoprecipitates are shown. In both immunoprecipitation assays, KLC1ser460ala bound approximately twofold more and KLC1ser460asp bound approximately twofold less calsyntenin-1 than KLC1wt. (B) Transfection of FLAG–KLC1 markedly increases KLC1 expression. Cells were transfected with empty vector (EV) or FLAG-tagged KLC1wt, KLC1ala or KLC1asp; total and transfected KLC1 was detected on immunoblots using anti-KLC1 and anti-FLAG antibodies as indicated. The samples were also probed for actin as a loading control. (C) siRNA-mediated knockdown of endogenous human but not transfected mouse FLAG–KLC1 in HEK-293 cells. Cells were co-transfected with either empty vector (EV) or FLAG–KLC1, and either control siRNA (Ctrl) or human-specific KLC1 siRNA as shown. Samples were probed on immunoblots for total KLC1 (KLC1) and transfected FLAG–KLC1 using the anti-FLAG antibody as indicated. The samples were also probed for actin as a loading control. (D) KLC1ser460ala binds approximately twice, and KLC1ser460asp binds approximately half, the calsyntenin-1 than KLC1wt following siRNA-mediated knockdown of endogenous KLC1. Cells were co-transfected with EGFP–calsyntenin-1 (calsyntenin) and human-specific KLC1 siRNA, and mouse FLAG–KLC1wt (KLC1wt), FLAG–KLC1ser460ala (KLC1ala) or FLAG–KLC1ser460asp (KLC1asp). KLC1 was immunoprecipitated using anti-FLAG antibody and the input and immunoprecipitate samples probed on immunoblots for KLC1, using the anti-FLAG antibody, and calsyntenin-1, using anti-GFP antibody.