Fig 6. Identification of proteins interacting with gelatin sepharose.

Proteins that bound tightly to gelatin sepharose were eluted with SDS sample buffer. (A-C) Images of Western blots are shown. Cell lysate and gelatin sepharose (gel-seph) elution represent the input cell lysate that was loaded onto gelatin sepharose and the eluted fractions from gelatin sepharose. The blot against β-tubulin is an input control. The blots with clear differences between WT and R222S HSP47 are shown. Blots against the other proteins are shown in S9A Fig. (D) Semi-quantitative Western blotting was performed for proteins that were reported to interact with gelatin sepharose. The protein signals of the control sample were set to 1 and compared to the patient sample. Data presented are means ± SEM and individual data points represent four or more independently prepared cell lysate (n>4). *: p<0.05, **: p<0.01 and ***: p<0.001 by unpaired t test. (E) Silver staining was performed for the eluted fractions from the gelatin sepharose. The arrowhead and asterisk (*) highlight the predicted migration of BiP and HSP47, respectively. ‘-‘ indicates the eluted fraction from gelatin sepharose mixed with TBS buffer instead of cell lysate as a blank.