Primary control and patient‐derived fibroblasts (Patient 1 a) were cultured in Ham’s F10 (Life Technologies, Paisley, UK) supplemented with 12% fetal calf serum.
To investigate intracellular procollagen transport in control and patient cells, cells were grown for 48–72 hr on glass coverslips and incubated in the presence of 167 µM (50 µg/ml) ascorbic acid for 30 min prior to fixation. Cells were fixed with 4% paraformaldehyde in PBS and processed for immunofluorescence using either only anti‐collagen IαI (Novus Biologicals NB600‐408, rabbit, 1:2,000) for analysis of the extracellular collagen, or anti‐collagen IαI in combination with the COPII marker anti‐Sec31A (BD Biosciences 612,350, mouse, 1:1,000) or the cis‐Golgi marker anti‐GM130 (BD Biosciences 610,823, mouse, 1:1,000) and imaged by confocal microscopy as described previously (McCaughey et al.,2016 ) using an Alexa‐Fluor‐anti‐rabbit‐568‐conjugated (Thermo Fisher Scientific) and/or Alexa‐Fluor‐anti‐mouse‐488‐conjugated secondary antibody, Prolong Diamond with DAPI for mounting and a Leica SP5 for analysis and image acquisition.
To investigate intracellular procollagen transport in control and patient cells, cells were grown for 48–72 hr on glass coverslips and incubated in the presence of 167 µM (50 µg/ml) ascorbic acid for 30 min prior to fixation. Cells were fixed with 4% paraformaldehyde in PBS and processed for immunofluorescence using either only anti‐collagen IαI (Novus Biologicals NB600‐408, rabbit, 1:2,000) for analysis of the extracellular collagen, or anti‐collagen IαI in combination with the COPII marker anti‐Sec31A (BD Biosciences 612,350, mouse, 1:1,000) or the cis‐Golgi marker anti‐GM130 (BD Biosciences 610,823, mouse, 1:1,000) and imaged by confocal microscopy as described previously (McCaughey et al.,