Fig 1.
Intracellular expression and extracellular secretion of MANF and CDNF in HEK293 cells.
(A) Schematic representation of the mouse MANF and CDNF expression constructs used in this study. SP indicates a signal peptide at the N-terminus of each protein. The cysteines are indicated by bars. The four C-terminal amino acids, RTDL and QTEL, putative ER localization signals at their C-termini are shown in capital letters. (B) Western blot analysis of wild-type and modified MANF and CDNF overexpressed in HEK293 cells. Twenty-four hours after transfection of each indicated construct into the cells, the culture medium was replaced with fresh serum-free DMEM, and the cells were incubated for an additional 12 h. The amounts of the indicated proteins in the cell lysate and culture medium were detected by western blot analysis using antibodies against Flag-epitope, MANF, CDNF and actin as described in the Materials and Methods. Representative data of three independent experiments were shown.
Fig 2.
Effects of mutant-Sar1 co-expression on the secretion of MANF and CDNF from HEK293 cells.
(A) Twenty-four hours after the transfection of SP-Flag-MANF or SP-Flag-CDNF with Sar1(H79G) or the empty vector (mock) into HEK293 cells, the culture medium was replaced with fresh serum-free medium and the cells were cultured for an additional 12 h. The amounts of MANF and CDNF in the cell lysate and culture medium were detected by western blot analysis as described in the Materials and Methods. Representative data of three independent cultures were shown. (B) Twenty-four hours after the transfection of SP-NL-MANF or SP-NL-CDNF with Sar1(H79G) or the empty vector (mock) into HEK293 cells, the culture medium was replaced with serum-free medium, and the cells were incubated for an additional 4 h. The culture medium (b, e) and cell lysate (c, f) from HEK293 cells expressing SP-NL-MANF or SP-NL-CDNF were collected. The luciferase activity in each sample was measured as described in the Materials and Methods. The values represent the mean ± SEM from nine independent cultures. The relative amounts of secreted SP-NL-MANF and SP-NL-CDNF in each case (a, d) were calculated from the data of their extracellular activities (b, e) and their intracellular activities (c, f), respectively. The data were analyzed by Student’s t-test to evaluate the effects of the co-expression of Sar1(H79G) on the luciferase activity. The values marked with an asterisk are significantly different from the value of the mock-transfected cells, respectively (p < 0.05).
Table 1.
The relative amounts of SP-NL-MANF and SP-NL-CDNF from HEK293 cells co-transfected with mock or Sar1(H79G).
Fig 3.
Effects of GRP78 co-expression on the secretion of MANF and CDNF from HEK293 cells.
(A) After the transfection of wtMANF or ΔCMANF (A-a) and wtCDNF or ΔCCDNF (A-b) with GRP78 or the empty vector (mock) into HEK293 cells, each indicated protein was detected as described in Fig 2. Representative data of three independent cultures were shown. The broken line represented the boundary line between wtCDNF and ΔCCDNF of the two lanes in the same immunoblotted membrane. (B) After the transfection of SP-NL-MANF (a, b, c) or SP-NL-CDNF (d, e, f) with GRP78 or the empty vector (mock), the luciferase activity of the culture medium (b, e) and cell lysate (c, f) from HEK293 cells expressing SP-NL-MANF or SP-NL-CDNF were measured and relative amounts of secreted SP-NL-MANF and SP-NL-CDNF in each case (a, d) were calculated as described in Fig 2. The values represent the mean ± SEM from six independent cultures. The data were analyzed by Student’s t-test to evaluate the effects of the co-expression of GRP78 on the luciferase activity. The values marked with an asterisk are significantly different from the value of the mock-transfected cells, respectively (p < 0.05).
Table 2.
The relative amounts of SP-NL-MANF and SP-NL-CDNF from HEK293 cells co-transfected with mock or GRP78.
Fig 4.
Effects of KDEL receptor1 co-expression on the secretion of MANF and CDNF from HEK293 cells.
(A) After the transfection of wtMANF or ΔCMANF (A-a) and wtCDNF or ΔCCDNF (A-b) with KDEL-R1 or the empty vector (mock) into HEK293 cells, the expression of indicated proteins was detected as described Fig 2. Representative data of three independent cultures were shown. (B) Twenty-four hours after the transfection of SP-NL-MANF (a, b, c) or SP-NL-CDNF (d, e, f) with KDEL-R1 or the empty vector (mock), the luciferase activity in each sample was measured and calculated as described in Fig 2. The values represent the mean ± SEM from six independent cultures. The data were analyzed by Student’s t-test to evaluate the effects of the co-expression of KDEL-R1 on the luciferase activity. The values marked with an asterisk are significantly different from the value of the mock-transfected cells, respectively (p < 0.05).
Table 3.
The relative amounts of SP-NL-MANF and SP-NL-CDNF from HEK293 cells co-transfected with mock or KDEL-R1.
Fig 5.
Intercellular localization of MANF and CDNF in COS7 cells.
(A, B) Forty-eight hours after transfection of SP-EGFP-MANF and SP-DsRed2-CDNF (A) or SP-DsRed2-MANF and SP-EGFP-CDNF (B) into COS7 cells, the cells were fixed and observed as described in Materials and Methods. Scale bar is 10 μm.
Fig 6.
Effects of mouse MANF and CDNF co-expression on the CRELD2 secretion from HEK293 cells.
(A) Schematic representation of the mouse MANF and CDNF expression constructs used in this study. SP indicates a signal peptide at the N-terminus of each protein. The cysteines are indicated by bars. The four or six C-terminal amino acids, RTDL, SARTDL, QTEL, YPQTEL, KTEL and HPKTEL, in each construct are shown in capital letter. After co-expression of wtCRELD2 with (B) wtMANF, ΔCMANF, wtCDNF or ΔCCDNF, (C) wtMANF, MANFYPQTEL or MANFQTEL, (D) wtCDNF, CDNFSARTDL or CDNFRTDL, (E) wtMANF, MANFHPKTEL or MANFKTEL, (F) wtCDNF, CDNFHPKTEL or CDNFKTEL, the indicated proteins were detected by western blot analysis as described in Fig 2. Representative data of three independent cultures were shown (B, C-a, D-a, E-a, F-a). The broken line represented the boundary line between the two lanes in the same immunoblotted membrane. (C-F b) Each of bar graphs shows densitometric analyses of the secreted CRELD2 as described in the Materials and Methods. Each value represents the mean ± SEM from 8 (C), 10 (D), 6 (E), 6 (F)-independent cultures. The values marked with an asterisk are significantly different from the values of the mock-transfected cells (p<0.05).