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. 2020 Sep 14;130(10):5523–5535. doi: 10.1172/JCI140155

Figure 3. RARRES1 is cleaved and released into the supernatants as a highly glycosylated soluble form.

Figure 3

(A) Cultured human podocytes were transiently transfected to express control EGFP or C-terminal V5-tagged RARRES1 (RARRES1-V5). Expression of RARRES1 was probed with V5 antibody in the cytoplasmic (C) and membrane (M) fractions in comparison with the total cell lysate (T). Pan-cadherin was used to confirm the membrane protein enrichment in the M fraction. (B) Total cell lysate (CL) or supernatant (Supe) from cultured podocytes expressing control EGFP or C-terminal V5-tagged RARRES1 (RARRES1-V5) was probed for RARRES1 expression using V5 antibody. (C) Total cell lysate or supernatant from cultured podocytes expressing control EGFP or N-terminal FLAG-tagged RARRES1 (FLAG-RARRES1) was probed for RARRES1 expression using FLAG antibodies. (D) Total cell lysate or supernatant from cultured podocytes expressing RARRES1-V5 with or without deglycosylation treatment was probed with V5 antibody. Arrowheads indicate shift in the RARRES1-V5 migration with or without deglycosylation. (E) Total cell lysate or supernatant from cultured podocytes expressing EGFP control, C-terminal V5-tagged RARRES1 with the deletion of aa 43–76 (Δaa43-76-V5), deletion of aa 43–55 (Δaa43-55-V5), or WT RARRES1 (RARRES1-V5) was probed with V5 and RARRES1 antibodies. (F) Supernatant from cultured podocytes expressing C-terminal V5-tagged RARRES1 with the deletion of aa 68–71 (ΔFFNF-V5), alanine substitution of individual amino acids of aa 68–71 as indicated in underlined A residue (AAAA-V5, AFNF-V5, FANF-V5, FFAF-V5, and FFNA-V5), aspartic acid substitution of aa 70 (FFDF-V5), or WT RARRES1 (RARRES1-V5) was probed for expression with V5 antibody. (G) Total cell lysate or supernatant from cultured podocytes expressing EGFP control, C-terminal V5-tagged RARRES1 (RARRES1-V5), or RARRES1 FFAF mutant (FFAF-V5) with or without the treatment of broad-spectrum MMP inhibitor Marimastat (50 μM) were probed with V5 antibody. (H) Total cell lysate, supernatant, or isolated ectosomes (Ecto) from cultured podocytes expressing EGFP control, C-terminal V5-tagged RARRES1 (RARRES1-V5), or RARRES1 FFAF mutant (FFAF-V5) was probed with V5 antibody.