Figure EV3. Cleavage of HuR by Leishmania membrane protease GP63.

• A
  Cleavage of HuR by Ld membrane‐derived soluble Leishmania antigen (SLA). The HuR cleavage is prevented by ortho‐phenanthroline (OPT). Protein equivalent amount of cell extract (100 μg) from RAW 264.7 cells were incubated with SLA (1 μg) in presence and absence of OPT for 30 min at 37°C, and level of HuR in treated cell lysate was determined by Western blot analysis. β‐Actin was used as loading control.
• B–E
  Cleavage of HuR by purified Gp63. The silver‐stained gel containing different fractions obtained during the dual steps purification of the GP63 from SLA derived from Ld membranes. Arrow mark the GP63 band (B). Increasing concentration of purified GP63 was incubated with fixed quantity of RAW 264.7 cell lysate and HuR cleavage reaction was done and HuR levels in each reaction were detected by Western blot (C). GP63 treatment cleaves HuR but it has no effect on β‐Actin or Alix present in the RAW 264.7 lysate. This denotes the specificity of this cleavage reaction (D). The HuR cleavage by purified GP63 can be blocked by OPT (E).
• F–I
  Schematic representation of GP63 entrapped liposome formation has been depicted (F). Purified GP63 was entrapped in specific liposome, and the liposome size and concentration were estimated using nanoparticle tracker. Size of the empty liposome was around 175.7 nm, and GP63 entrapped liposome was around 191.8 nm. Concentration of empty liposome was estimated to be around 6 × 10⁸ particles/ml, and GP63 entrapped liposome was in the range of 6 × 10⁷ particles/ml (G). RAW 264.7 cells were treated with 1 μg of GP63 entrapped liposome and incubated for 6 and 16 h, after which cells were harvested to check for HuR levels. Visible reduction in HuR levels was observed along with unchanged Ago2 levels, suggesting a specific cleavage action on HuR. Empty liposome treatment was used as a control (H). GP63‐containing liposomes were used to cleave HuR in RAW 264.7 cells. C1 and C2 are control set with empty liposome treated, and gp1, gp2 and gp3 are GP63 liposome‐treated cells. HuR and Dicer1 levels were checked in cell lysates. Dicer1 has been previously reported to be cleaved by GP63 and hence was used as positive control. β‐Actin was used as loading as well as negative control (I). Positions of molecular weight markers are marked and shown in the Western blots used in different panels.

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