Validation of processing defects in other giantin mutant RPE1 lines. (A) Immunoblot of lystates from WT and giantin exon 13 KO RPE1 clones probed for giantin (polyclonal against full-length protein) and tubulin (housekeeping). (B) Widefield maximum-projection images of WT and giantin KO RPE1 cell clones immunolabeled for the N-terminus (N-term) of, and full-length (FL) giantin; ER exit sites (Sec31); and the Golgi (GRASP65) as indicated. Scale bar = 10 µm. (C) Immunoblots of secretion assays performed on WT and giantin KO clones stably expressing GFP-COL1A1 and untransfected giantin KO cells (unTF). Blots show the medium (M) and lysate (L) fractions of each cell population immunoblotted for GFP, SBP, and pro-α1(I) N-propeptide as indicated. (D) Maximum-projection widefield images of GFP signal in giantin KO clones stably expressing GFP-COL1A1 and immunolabeled for pro-α1(I) N-propeptide (magenta). Scale bar = 10 µm.
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