antibodie mfsd2a  (Bioss)

Journal: Oncotarget

Article Title: Cytotoxic stress induces transfer of mitochondria-associated human endogenous retroviral RNA and proteins between cancer cells

doi: 10.18632/oncotarget.21606

Figure Lengend Snippet: Panel A: controls. A1 : Mitochondrial Cox IV was detected in whole cell extracts, nuclear and mitochondrial fractions. A2 : lamin A & C markers were detected only in nuclear fractions indicating the absence of nuclear contaminations in the mitochondrial fraction except for cytoplasmic preparations. Panel A3 reflects the content of syncytin 2 (HERV-FRD 1 ) in all sub-cellular preparations with remarkable expression in the mitochondrial fraction of U87 RETO cells. Panel A4 : detection of the receptor for syncytin 2, MFSD2 in subcellular fractions except for cytoplasmic preparations. Panel B minor expression of pro-apoptotic proteins like BAD and BAX in U87 RETO cells. In contrast, anti-apoptotic proteins like Bcl-2 and Bcl-xL were found strongly expressed. (For comparison of cytoplasmic vs. whole cell distributions of BAX in untreated vs. treated cells, showing no difference upon etoposide-incubation, see .) Panel C : controls: isolated mitochondria with mitochondrial markers MFN1 and MFN2 (positive control) almost without extra-mitochondrial membrane proteins like ABCG2 and lamin A+C (negative controls). Panel D1 : expression of different HERV proteins in the mitochondrial fraction of U87 RETO cells. For syncytin 1 (HERV-W E1 , upper bands) the 53 kDa surface protein and an additional splicing variant below is shown. In addition, the 24 kDa band reflects the transmembrane protein of syncytin 1. For syncytin 2 the 24 kDa protein was not detectable. HERV-V 3-1 was detectable as the expected single band at 32 kDa. Panel D2 : analysis of intracellular distribution of syncytin 1 in untreated U87 RETO cells (control, left lanes) vs. treated U87 RETO cells after incubation with 5 μg/ml etoposide for 10 days. HERV proteins were found enhanced in the mitochondrial fraction upon etoposide stress. Comparable results were obtained for syncytin 2, see . Panel E : after incubating U87 RETO cells with 5 μg/ml etoposide for 10 days, receptors for syncytin 1 and 2 were also clearly detectable in mitochondrial fraction (see also panel A4 ). For SCL1A5, the endogenous protein (53 kDa) and various glycosylated forms were detected. The receptor for syncytin 2, MFSD2, was also detectable as a single band. The figure is representative of at least n = 3 independent experiments.

Article Snippet: Primary antibodies were purchased as follows: anti-HERV-WE 1 (syncytin 1) and anti-HERV-FRD 1 (syncytin 2) from Bioss, USA (Cat. No. bs2962R and bs15466R) and Biorbyt, UK (Cat. No. orb111912); anti-lamin A+C from Novus Biologicals, USA (Cat. No. EPR4100); Cox IV (Cat. No. 11967), ABCG2 (Cat. No. 4477), MFN1 (Cat. No. 13196), MFN2 (Cat. No.9482), Bcl-2 (Cat. No. 2870), Bcl-X L (Cat. No.2764), BAD (Cat. No.9239) and BAX (Cat. No.5023) primary antibodies from Cell Signaling Technology, (USA); polyclonal antibodies targeting SLC1A5 (Cat. No. bs-0473R) and MFSD2A (Cat. No. bs-6073R) from Bioss, USA; conjugated secondary antibodies from Cell Signaling (USA) and Jackson ImmunoResearch Europe Ltd. (Suffolk, UK).

Techniques: Expressing, Incubation, Isolation, Positive Control, Variant Assay

Journal: Oncotarget

Article Title: Cytotoxic stress induces transfer of mitochondria-associated human endogenous retroviral RNA and proteins between cancer cells

doi: 10.18632/oncotarget.21606

Figure Lengend Snippet: Mitochondrial samples were dual labeled for Cox IV and different HERV-proteins. Panel A depicts the expression of syncytin 1 (HERV-WE 1, A1 ), syncytin 2 (HERV-FRD 1 , A2 ), their respective receptors SLC1A5 (A3) and MFSD2 (A4) in U87 cells, which were used as source of mitochondria. The total HERV-protein expression in whole U87 cells was set to 100 %, and the relative amount of HERV-proteins associated with the mitochondrial fraction was given in relationship to the total HERV-protein amount in whole cells. Panel B depicts the measurement of different HERVs in the mitochondrial fraction of these U87 glioblastoma cells. The Cox IV- positive gated populations represent more than 94 % (picture B2 ). Picture B3 represents the negative control (secondary antibody). Both syncytin 1 and syncytin 2 ( B4 & B5 ) were found to be highly expressed, accounting for more than 98 %. HERV-V 3-1 ( B6 ) was detected in more than 67 % of the Cox IV positive populations. Plots are representative of at least n = 3 experiments.

Article Snippet: Primary antibodies were purchased as follows: anti-HERV-WE 1 (syncytin 1) and anti-HERV-FRD 1 (syncytin 2) from Bioss, USA (Cat. No. bs2962R and bs15466R) and Biorbyt, UK (Cat. No. orb111912); anti-lamin A+C from Novus Biologicals, USA (Cat. No. EPR4100); Cox IV (Cat. No. 11967), ABCG2 (Cat. No. 4477), MFN1 (Cat. No. 13196), MFN2 (Cat. No.9482), Bcl-2 (Cat. No. 2870), Bcl-X L (Cat. No.2764), BAD (Cat. No.9239) and BAX (Cat. No.5023) primary antibodies from Cell Signaling Technology, (USA); polyclonal antibodies targeting SLC1A5 (Cat. No. bs-0473R) and MFSD2A (Cat. No. bs-6073R) from Bioss, USA; conjugated secondary antibodies from Cell Signaling (USA) and Jackson ImmunoResearch Europe Ltd. (Suffolk, UK).

Techniques: Labeling, Expressing, Negative Control

Journal: Oncotarget

Article Title: Cytotoxic stress induces transfer of mitochondria-associated human endogenous retroviral RNA and proteins between cancer cells

doi: 10.18632/oncotarget.21606

Figure Lengend Snippet: Panel A 1 & 2 : positive controls, mitochondria were previously isolated from U87 RETO cells after cytotoxic stress with etoposide (as described above) and labeled with red MitoTracker ® . Endogenous mitochondria of the U87 host-cells were stained with green MitoTracker ® . After 24h-incubation without further cytotoxic stress, cellular uptake of prepared “red” mitochondria into cancer cells was clearly detectable. Panels 3-6 : experiments were performed under the same conditions as described for panels A 1 & 2 . Panels A 3 & 4 : mitochondria isolated from U87 RETO were blocked with both anti-syncytin 1 (HERV-W E1 ) and anti-syncytin 2 (HERV-FRD 1 ) blocking antibodies and the uptake of the labeled mitochondria was monitored after 24 hours. Panels A 5 & 6 : mitochondria isolated from U87 RETO were blocked with both anti-SLC1A5 and anti-MFSD2 blocking antibodies and the uptake of the labeled mitochondria was monitored after 24 hours. These results indicate that syncytin 1 and 2 as well as their receptors play a substantial role in cellular uptake of mitochondria, since the uptake of exogenous mitochondria were less perceived after blockage of these proteins. Panel B : cartoon representation of the proposed mechanisms of mitochondria entrance into U87 cells. Mitochondria from U87 RETO etoposide resistant cells were isolated and labeled with MitoTracker© red and with anti syncytin 1 or anti syncytin 2 antibodies and exogenously applied to U87 cells. The blockage of both syncytins impedes the uptake of free mitochondria.

Article Snippet: Primary antibodies were purchased as follows: anti-HERV-WE 1 (syncytin 1) and anti-HERV-FRD 1 (syncytin 2) from Bioss, USA (Cat. No. bs2962R and bs15466R) and Biorbyt, UK (Cat. No. orb111912); anti-lamin A+C from Novus Biologicals, USA (Cat. No. EPR4100); Cox IV (Cat. No. 11967), ABCG2 (Cat. No. 4477), MFN1 (Cat. No. 13196), MFN2 (Cat. No.9482), Bcl-2 (Cat. No. 2870), Bcl-X L (Cat. No.2764), BAD (Cat. No.9239) and BAX (Cat. No.5023) primary antibodies from Cell Signaling Technology, (USA); polyclonal antibodies targeting SLC1A5 (Cat. No. bs-0473R) and MFSD2A (Cat. No. bs-6073R) from Bioss, USA; conjugated secondary antibodies from Cell Signaling (USA) and Jackson ImmunoResearch Europe Ltd. (Suffolk, UK).

Techniques: Isolation, Labeling, Staining, Incubation, Blocking Assay