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. 2020 Jan 1;219(2):e201908182. doi: 10.1083/jcb.201908182

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© 2020 Chen et al.

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Figure 5. — RTN3 protects ER membrane integrity during ER exit of misfolded Akita proinsulin aggregates to the lysosome for degradation. (a) COS-7 cells expressing FLAG-NHK or Akita-Myc were transfected with either scrambled or Beclin-1 siRNA, fixed, permeabilized, and stained with FLAG/Myc and RTN3 antibody. Images were taken on a Zeiss LSM 800 confocal microscope and Airyscan processed. Scale bars, 10 µm. (b) Quantification of the percentage of Beclin-1 knockdown cells containing Akita-Myc or FLAG-NHK puncta. Values represent means ± SD from three independent experiments. (c) The detergent-insoluble fraction derived from RTN3-depleted COS-7 cells expressing FLAG-NHK (first panel) or Akita-Myc (second panel) were layered over a 10% to 50% discontinuous sucrose gradient and centrifuged. Each fraction was collected, subjected to SDS-PAGE, and immunoblotted with antibody against FLAG or Myc. Purified SV40 was also layered over a 10–50% sucrose gradient and centrifuged. Each fraction was collected, subjected to SDS-PAGE, and immunoblotted with the antibody against VP1 (third panel). (d) COS-7 cells expressing GFP-NHK or GFP-Akita were transfected with scrambled, RTN3, or RTN4 siRNA. Cells were then subjected to the protease protection assay as in Fig. 4 d. (e) The graph represents the intensity of the BiP and GFP-NHK/GFP-Akita band in c. The band intensity was quantified by ImageJ using scans of films after ECL. Results represent the mean of three independent experiments. A two-tailed t test was used. Error bars, ± SD.