Amicon ultra centrifugal filters 100 kb

Cells were seeded in a 96-well black plate (Sarstedt, Nümbrecht, Germany) at 5000 cells/well in three replicates until con uence. The media was then changed to differentiation medium. Cells were treated with the corresponding drug for the indicated time. CT-L, C-T and T-L activity was assayed by chemiluminiscence using the Proteasome-Glo™ 3-Substrate System cell based assay (Promega, Madison, WI, USA ) and the plate was read using Victor 3v Multilabel Plate Reader (Perkin Elmer, Waltham, MA, USA).
Thrombospondin-1 Enzyme-Linked Immunosorbent Assay (ELISA)
Immortalized myoblasts from the dysferlinopathy patient were seeded at 5000 cells/cm 2 and expanded until con uence. The media was then changed to differentiation medium to form myotubes. After treatment with proteasome inhibitors together with or without EB1089, the media was removed and added 1 ml of basal DMEM (Lonza) was added to the culture for 24 hours. Cell culture supernatants were concentrated using Amicon Ultra Centrifugal Filters 100 kb (Merck Millipore, Darmstadt, Germany). TSP-1 present in the culture media was detected using the human TSP-1 Immunoassay (Quanti-Kine ELISA, R&D Systems, Minneapolis, MN), following the manufacturer's instructions. The detection limit of the assay was 0.355 ng/mL.

Cells were seeded in a 96-well black plate (Sarstedt, Nümbrecht, Germany) at 5000 cells/well in 3 replicates until con uence. The media was then changed to differentiation medium. Cells were treated with the corresponding drug for the indicated time. The CT-L, C-T and T-L activity was assayed by chemiluminiscence using the Proteasome-Glo™ 3-substrate System cell based assay (Promega, Madison, WI, USA ) and the plate was read using Victor 3v Multilabel Plate Reader (Perkin Elmer, Waltham, MA, USA).
Thrombospondin-1 Enzyme-Linked Immunosorbent Assay (ELISA)
Immortalized myoblasts from the dysferlinopathy patient were seeded at 5000 cells/cm 2 and expanded until con uence. The media was then changed to differentiation medium to form myotubes. After treatment with proteasome inhibitors together with or without EB1089, we removed the media and added 1 ml of basal DMEM (Lonza) to the culture for 24 hours. Cell culture supernatants were concentrated using Amicon Ultra Centrifugal Filters 100 kb (Merck Millipore, Darmstadt, Germany). TSP-1 present in the culture media was detected using the human TSP-1 Immunoassay (Quanti-Kine ELISA, R&D Systems, Minneapolis, MN), following the manufacturer's instructions. The detection limit of the assay was 0.355 ng/mL.