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Fig. 3. Sdc1-null keratinocytes produce a matrix distinct from that made by wt keratinocytes. (A) Sirius Red dye binding assay to measure collagen accumulation in the wt and Sdc1-null cultures shows that the day after the keratinocytes had been tracked, Sdc1-null keratinocyte cultures had accumulated significantly less collagen per keratinocyte compared with wt keratinocytes. (B) Matrix preparations identical to those used in the experiments described in Fig. 2D were extracted as described, normalized and run on 4-20% gels that were silver-stained. High-molecular-mass proteins accumulated in the matrices, but there were no major differences between the amount of high-molecular-mass matrix deposited by the wt and Sdc1-null keratinocytes. The lower molecular mass bands are keratins, which stick non-specifically to the matrix after keratinocytes are lysed. The control extract shows those proteins deposited on wells which remain after ammonium hydroxide treatment of wells that had been coated with FN-CNI and fed serum-containing medium. (C) Immunoblots of the same matrix preparations shown in B normalized by cell count and probed for LN-332 using the J18 antibody showed similar patterns for LN-332 unprocessed (unp) and processed (prc) 
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2 chains for matrices deposited by wt and Sdc1-null keratinocytes. (D) Immunofluorescence microscopy using the J18 antibody on matrix preparations from cultures of wt and Sdc1-null keratinocytes. (a,d) 20x images taken of wt and Sdc1-null keratinocytes; asterisks indicate elongated clear areas lacking LN-332 surrounded by areas positive for LN-332. In b and e, asterisks indicate regions shown at higher resolution. (c and f) Additional high-resolution images of wt and Sdc1-null keratinocyte matrices, respectively. Arrows in b,c,e,f indicate ordered streaks that are more prominent in Sdc1-null matrix than in wt matrix; arrowheads indicate amorphous cloud-like staining present in wt matrix but largely absent in Sdc1-null keratinocytes. Bar, 6 µm for a,d, 2 µm for b,c,e,f.
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