Human IL‑22BP Antibody

Detection of Human IL-22BP/IL22 RA2 by Western Blot

Differential secretion, intracellular retention and degradation by the proteasome of IL-22BP isoforms. Data refer to HEK293 cells transiently transfected for 24 h unless otherwise specified. (A) HEK293 cells were transiently transfected with expression vectors encoding IL-22BPi1, BPi2, or BPi3. Intracellular and secreted IL-22BP protein levels were measured by WB (FLAG Ab) and ELISA, respectively. Transfection efficiency was measured by IL22RA2 RT-qPCR relative to the housekeeping gene GAPDH (mean ± SEM; n = 3). (B) Confocal microscopy of IL-22BP isoforms (green, IL-22BP Ab 4 with either ERp72 or trans-Golgi marker TGOLN2 (red) in transfected HEK293 cells; cells were counterstained with DAPI. (C) Secreted recombinant IL-22BPi2 is not detected by WB in unconcentrated conditioned media (CM). Various cytokine expression vectors, as indicated, were transiently transfected and CM were collected and immunoblotted for FLAG. (D) Detection of IL-22BP isoforms in cell lysates (CL) and acetone-precipitated CM (AP) by FLAG Ab. Secreted IL-22BPi2 was treated with PNGase F or Endo H. (E) Intracelullar isoforms of IL-22BP were treated with PNGase F or Endo H and detected by FLAG Ab. (F) HEK293 cells were transfected for 24 h with expression vectors for either IL-2 or IL-2EX4. The latter was generated by subcloning exon-4 from IL22RA2v1 into the open reading frame of IL-2 through its unique Xba1 position. Detection of resulting proteins in AP, CL, CM by FLAG Ab. (G) HEK293 cells were transiently transfected with the same expression plasmids as in (A) or IL-2EX4; 24 h later, cells were treated with a mix of proteasome inhibitors (PI) (5 μM lactacystin, 5 μM MG132, and 1 mM epoxomicin), and 18 h later cells were lysed and immunoblotted for FLAG using GAPDH as a loading control. All data are from at least 3 independent experiments. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/30619294), licensed under a CC-BY license. Not internally tested by R&D Systems.