Skip to main content
. 2020 Nov 9;9:e62506. doi: 10.7554/eLife.62506

Figure 5. Differential protein distribution into syt7 and syt9 granule subpopulations.

(A) Palmitic acid incorporates more efficiently into INS 832/13 cell-derived syt7 than into syt9 granules. Fluorescence spectra (left) of purified insulin granules with incorporated Click-iT palmitate solubilized in 1% Triton-X and then labeled with Alexa647 (by an Azide-Alkyne Click-iT reaction). The spectra are those of granules before (green) and after immunodepletion with syt antibodies (red, depletion using anti-syt7; blue, depletion using anti-syt9). Quantification of fluorescence change in control and syt7 or syt9 granules (right). The results are from three separate purifications of insulin granules. (B) Distribution of granule proteins between syt7 and syt9 granules that were derived from GRINCH cells. Data show the fraction of the total of each protein associated with syt7 (blue) vs syt9 (red) granules as determined from quantitative western blotting (Figure 5—figure supplement 1); error bars are standard deviation from three independent samples. For statistics see Figure 5—source data 1. (C) Model summarizing the compositions of syt7 (blue) and syt9 (red) insulin granules.

Figure 5—source data 1. Significance values of protein contents between different immunodepleted granules obtained from Figure 5B and Figure 5—figure supplement 1.

Figure 5.

Figure 5—figure supplement 1. Western blots for proteins associated with GRINCH cell-derived insulin granules before immunodepletion (left lanes) and after immundepletion of intact granules using antibodies against syt7 (middle lanes) or syt9 (right lanes).

Figure 5—figure supplement 1.

Samples were divided into three equal parts: control (unaltered) and each of the other two immunodepleted. All three were solubilized in SDS-loading buffer and western blotting was performed on an equal fraction of the total of each sample. The three bands in the GFP blot are from top to bottom: proinsulin-GFP, conversion intermediate, C-peptide-GFP. The results shown were reproduced in three completely independent experiments.