Figure 5.

Identification of the essential sequence. (a) The alpha helixes and loop domains of the J-domain fragment sequence are indicated by brackets. As shown in the alignment, the original J-domain fragment sequence is 61 amino acids in length. Truncated J-domain fragment sequences used in this assay were depicted (Jdel^erdj3-Jdel6^erdj3). (b) The original sequence (J^erdj3) and deletion polypeptides derived from a Erdj3 J-domain fragment sequence (Jdel^erdj3 ~ Jdel6^erdj3) were linked to protein A, forming a fusion protein that was co-expressed with a V5-tagged IL13Rα2-Fc target protein in transfected cells. The level of expression of the V5-tagged target protein in culture media was determined in an immunoblot assay using an anti-V5 antibody, and expression of fusion proteins carrying an original or truncated J-domain sequence was detected in cell lysate by western blot analysis using an anti-Flag antibody. All transfectants were co-transfected with a reporter plasmid expressing GFP. The cell lysate membrane was subsequently probed with an anti-GFP antibody (as a transfection control) (bottom panel). (c) The corresponding sequences from various J-domain proteins are aligned. The corresponding sequence derived from other J-domain proteins was conjugated to protein A to make a fusion protein, which was co-expressed with a V5-tagged IL13Rα2-Fc target protein in transfected cells. The V5-tagged IL13Rα2-Fc target proteins in both culture media and cell lysates were analyzed by western blot analysis using an anti-V5 antibody, and the fusion proteins incorporating the corresponding sequences derived from DnaJ protein were detected in cell lysate with an anti-Flag antibody. The cell lysate membrane was subsequently probed with anti-GFP antibody as a transfection control (bottom panel). (d) For mutation analysis, the second and third amino acids in the corresponding sequence from Erdj4 (IKKAFHKLAMKY) were substituted for alanine and conjugated to protein A (MT1: IAAAFHKLAMKY). MT2 (IKKAAHKLAMKY) and MT3 (IKKAFHALAMKY) have a point mutation at 5^th and 7^th amino acid, respectively, and MT4 has both mutations at the same position (IKKAAHALAMKY). The fusion proteins with native or mutated sequence (Jdel5^erdj4-TD^A or Jdel 5^erdj4MT-TD^A) were expressed with a V5-tagged IL13Rα2-Fc target protein in HEK293 cells. The protein levels of the V5-tagged IL13Rα2-Fc target protein in both transfected cells and culture media were analyzed by western blot analysis using an anti-V5 antibody. The protein-A fusion proteins in transfected cells were analyzed using an anti-Flag antibody. The cell lysate membrane was subsequently with anti-GFP antibody as a transfection control (bottom panel).