Figure 4.

Figure 4.

Procollagen processing is defective in giantin KO RPE1 cells. (A) Representative images of WT and giantin KO cells expressing GFP-COL1A1 or GFP alone as indicated and immunolabeled for pro-α1(I) (COL1A1, magenta). Cells are not expressing a RUSH hook. Nuclei are stained with DAPI (blue). Images are maximum-intensity projections of widefield z-stacks. Scale bars = 10 µm. (B–F) Immunoblots of secretion assays showing the medium (M) and lysate (L) fractions of WT and giantin KO cell cultures immunoblotted for GFP and tubulin (B), SBP and tubulin (C), pro-α1(I) C-propeptide (LF41 antibody) and tubulin (D), pro-α1(I) N-propeptide (LF39 antibody; E), and pro-α1(I) and GAPDH (F). Black arrows indicate full-length procollagen, and red arrows highlight N-propeptide bands. (G) Maximum-intensity projection widefield z-stacks of WT and giantin KO cells stably expressing GFP-COL1A1 immunolabeled for the pro-α1(I) N-propeptide (LF39 antibody). Scale bars = 10 µm. Ab, antibody.