Fig 2: Preincubation with untagged human embryonal kidney cells 293 (HEK293) cell-derived progranulin (PGRN) does not interfere with tumor necrosis factor (TNF)-tumor necrosis factor receptor-1 (TNFR1) and TNF–TNFR2 interaction in cell-free and cellular binding studies. (A) HEK293 cells were transfected with empty vector (EV) or an expression plasmid encoding non-tagged PGRN. The indicated volume of supernatants (SNs) and cell lysates derived from these transfectants along with recombinant PGRNAdi (30, 15, and 7.5 ng) as standard were analyzed by Western blotting for the presence of PGRN and estimation of PGRN concentration. PGRN concentrations reached approximately 10,000 ng/ml in the SN of PGRN transfected cells (PGRNSN) and approximately 30,000 ng/ml in the corresponding cell lysate (PGRNlys). There was no detectable endogenous PGRN expression neither in the SN (EVSN) nor the lysate (EVlys) of EV-transfected cells. (B,C) TNFR2-Fc or, as a control for unspecific binding, IgG1 was immobilized to black enzyme-linked immunosorbent assay plates. Lysates (B) and SNs (C) of PGRN and EV-transfected cells [PGRNlys and EVlys (B), PGRNSN and EVSN (C)] were added for 1 h before the specific binding of 50 ng/ml and GpL-TNF was determined in triplicates. Where indicated immobilized TNFR2-Fc was pretreated for 1 h with 2,000 ng/ml sTNF or 20 µg/ml of a neutralizing TNFR2-specific antibody (aTNFR2). (D) HEK293 transfectants expressing TNFR1 or TNFR2 along with control HEK293 cells transfected with EV were preincubated for 1 h with pure PGRNSN, pure EVSN, and pure EVSN with and without supplementation with 10,000 ng/ml sTNF. After preincubation, cells were incubated in triplicates with 10 ng/ml GpL-TNF at 37°C for 1 h and finally cell-bound GpL activity was determined. ***p < 0.0001; **p < 0.001.

Fig 3: Gaussia princeps luciferase (GpL)-tumor necrosis factor (TNF) binding to cells with endogenous coexpression of tumor necrosis factor receptor-2 (TNFR2) and progranulin (PGRN). (A) Human embryonal kidney cells 293 (HEK293) cells were transfected (first transfection) with empty vector (EV) or an expression vector encoding TNFR2, where the TNF receptor associated factor 2 (TRAF2) binding site has been replaced by yellow fluorescence protein (YFP) (TNFR2). The following day, transfecfed cells were split into four aliquots which were transfected a second time (second transfection) with expression plasmids encoding PGRN, membrane TNF (memTNF), soluble Flag-tagged TNF (F-TNF), or EV. After an additional day, aliquots of 30,000 cells (P) and 15 µl SN (S) were analyzed by Western blotting with anti-PGRN, anti-TNF, and anti-Flag along with 100 ng PGRNAdi and 100 ng purified untagged soluble TNF (sTNF). (B) Equilibrium binding studies were performed with the indicated concentrations of GpL-TNF. Specific binding of GpL-TNF in the presence of PGRN (second transfection PGRN), membrane TNF (second transfection memTNF), and Flag-TNF (second transfection F-TNF) or the absence of an potential modulator (second transfection EV) was obtained by subtracting unspecific binding values (first transfection EV) from the corresponding total binding values (first transfection TNFR2). Specific binding values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.

Fig 4: Gaussia princeps luciferase (GpL) fusion proteins of progranulin (PGRN) show no relevant binding to tumor necrosis factor receptor-1 (TNFR1) or TNFR2. (A) Human embryonal kidney cells 293 (HEK293) cells were transiently transfected with expression plasmids encoding GpL-PGRN (GpL-PGRN), PGRN-GpL (PGRN-GpL), or empty vector (EV). GpL-PGRN concentrations in supernatants (SNs) and cell lysates were determined by help of a GpL fusion protein of known concentration. SNs and cell lysates, containing approximately 100 ng PGRN-GpL or GpL-PGRN along with 100 ng PGRNAdi, were subjected to Western blotting with a PGRN-specific antibody to verify the integrity of the PGRN GpL fusion proteins. (B) TNFR1-Fc, TNFR2-Fc or, as a control for unspecific binding, hIgG1 were immobilized to black enzyme-linked immunosorbent assay plates. Lysates and SN of the GpL-PGRN (GpL-PGRNlys and GpL-PGRNSN) and PGRN-GpL (PGRN-GpLlys and PGRN-GpLSN) transfected cells and GpL-tumor necrosis factor (TNF) were added for 1 h and binding was determined in triplicates. (C) TNFR1 and TNFR2 expressing transfectants (total binding) and EV-transfected HEK293 cells (non-specific binding) were subjected to equilibrium binding studies with the indicated GpL fusion proteins. Specific binding (= total - non-specific binding) values were fitted by non-linear regression analysis to a single binding site type of interaction by help of the GraphPad Prism 5 software.

Fig 5: Specificity of PGRN antibodies and detection of multiple endogenous GRNs. A, Whole-cell lysates from HAP1 WT and HAP1 PGRN KO cells, either reduced (+TCEP) or nonreduced (-TCEP), were analyzed for PGRN antibody specificity by immunoblot. The bottoms of the blots (<15 kDa) were exposed longer to reveal levels of endogenous GRNs. B, Whole-cell lysates from HEK293T WT and N-TAP PGRN stable overexpressing (PGRN) cells were analyzed as in A by immunoblot. C, Representative immunofluorescent images of HAP1 PGRN KO and WT cells stained with the corresponding PGRN antibodies as in A and B. Scale bar, 10 µm.