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. 2018 Dec 17;38(2):e99847. doi: 10.15252/embj.201899847

Figure EV5. FAM134B‐CANX interaction is not modulated by PC.

Figure EV5

  • A
    HeLa (Kyoto) cells were transfected with empty vector control, FAM134B‐HA WT or mutant constructs as indicated. Complexes were immunoisolated with HA‐magnetic beads, separated by Western blot and visualized with antibodies against CANX, FAM134B and β‐actin as control. 5% of the input is shown. Western blots are representative of three independent experiments.
  • B, C
    WT and Canx −/− MEFs were transiently transfected with RFP‐LC3 and WT GFP‐FAM134B. Representative immunofluorescence, the insets show higher magnification (x3.62) and single colour channels of the boxed area. Scale bar = 10 μm. Quantification of AVs positive for RFP‐LC3 (red) containing GFP‐FAM134B (green) expressed as % of total LC3, FAM134B and FAM134B+LC3 per cells (mean ± SEM). n = 12 cells counted per condition; three independent experiments.
  • D, E
    Addition of HaloTag does not perturb trafficking of PC2 molecules. (D) Pulse chase of U2OS cells transfected with Mifepristone inducible HALO‐PC2. Cells were pulsed for 20 min with HALO ligand (568, TMR, red) then chased in medium containing HALO ligand (Far red, blue) for 0, 15, 30 min. Images show that after 30 min, the majority of TMR‐bound HALO was secreted. The insets show higher magnification (left = x2.56, middle = x3.31, right = x6.79) and single colour channels of the boxed area. Scale bar = 10 μm. (E) Scanning confocal analysis of U2OS transfected with HALO‐PC2, treated for 9 h with 100 nM BafA1 in the presence of TMR (red) and immunolabelled for LAMP1 (488, green). Nuclei were stained with Hoechst (blue). The insets show higher magnification (x4.52) and single colour channels of the boxed area. Scale bar = 10 μm.

Source data are available online for this figure.