Figure 5.

The cytosolic PRD of SHISA5 was required for the inhibitory role of SHISA5 at the contact. (A) Left panel: schematic diagram of the orientation of SHISA5 within the ER membrane. Right panel: domain structures of SHISA5 mutants used for analysis. (B, C) WT and SHISA5 KO HeLa cells were transfected with control empty, SHISA5-DsRed, TMPRD-SHISA5-DsRed, or TMCRD-SHISA5-DsRed plasmid (red) for 48 h. (B) LC3B puncta were immunostained and shown in the right panel. The numbers of LC3B puncta in each cell was quantified and shown in the left panel (n = 11–17). (C) Right: representative images of PLA between SEC16A and WIPI2 are shown, along with negative controls incubated with antibody against either SEC16A or WIPI2 alone. Left: the PLA signals in the indicated samples were quantified using ImageJ plugins (n = 10–15). See Figure S5A for the representative images of PLA before inverting the image display. (B) and (C) are derived from three and two independent experiments, respectively. The error bars indicate SEMs. All scale bars: 10 μm.