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. 2019 Feb 7;20(3):e46449. doi: 10.15252/embr.201846449

Figure EV3. SPPL2c cleaves selected tail‐anchored (TA) proteins.

Figure EV3

  1. Ectopically expressed murine SPPL2c also cleaves human heme oxygenase 1 (HO‐1). HEK293 cells were transiently transfected with human N‐terminally FLAG‐tagged HO‐1 (FLAG‐hHO‐1) alone or in combination with active or inactive (D/A) murine SPP or SPPL2c. Western blot analysis was performed with anti‐FLAG. SPP was detected with a polyclonal serum detecting the overexpressed murine and the endogenous human (h/m) SPP. SPPL2c was visualised with the antiserum against the C‐terminus of the protein. To control for equal protein loading, actin was visualised. *, non‐specific band.
  2. All TA proteins analysed in Fig 2 co‐localise with SPPL2c. HeLa cells were transiently transfected with N‐terminally HA‐tagged Ube2J1, CYB5A, RAMP4‐2 or RAMP4 together with inactive (D/A) murine SPPL2c fused to a Myc epitope at its C‐terminus. Substrates and the inactive proteases were visualised with anti‐HA and anti‐Myc, respectively, in conjunction with fluorophore‐conjugated secondary antibodies. Scale bars, 10 μm.
  3. Substrate specificity of SPPL2c Isoform B is similar to isoform A. The indicated proteins were transiently expressed in HEK293 cells either alone or together with murine SPPL2c isoform B (IsoB). The expressed substrate candidate proteins were detected with anti‐HA. Expression of SPPL2c was confirmed with anti‐SPPL2c directed against an N‐terminal epitope. Cofilin was used to confirm equal protein loading.

Source data are available online for this figure.