Mouse anti transferrin receptor antibody

Protein samples were separated by SDS-PAGE [37 (link)] and subsequently transferred onto PVDF membranes (GE Healthcare). Membranes were blocked with 3% bovine serum albumin and incubated with the appropriate primary antibody (Ab). HA-tagged ubiquitin was visualized using an HA-reactive monoclonal antibody (mAb) directly conjugated to horse radish peroxidase (HRP) (Roche, Basel, Switzerland). FLAG-ALIX was detected by an anti-FLAG mAb directly conjugated to HRP (Sigma, St. Louis, MO,USA). Gag was detected by a rabbit Ab recognizing p24 (Seramun, Heidesee, Germany). The anti-mouse and anti-rabbit secondary antibodies coupled to HRP were obtained from Dianova (Dianova, Hamburg, Germany). Protein bands were quantified using AIDA (Raytest, Straubenhardt. Germany). For the linkage specific staining of polyubiquitin the rabbit mAb Apu2 (Millipore, Darmstadt, Germany) was used for the detection of K48-linked polyubiquitin and the mouse mAb HWA4C4 (Enzo, Farmingdale, NY, USA) for K63-linked polyubiquitin. Recombinant polyubiquitin chains either linked via the internal lysine 48 or 63 (Boston Biochem, Cambridge, MA, USA) served as specificity control. The mouse anti-Transferrin receptor antibody was purchased from Life Technologies and the anti-β-actin antibody from Sigma.

Protein samples were separated by SDS-PAGE [40 (link)] and subsequently transferred onto PVDF membranes (GE Healthcare). Membranes were blocked with 3% bovine serum albumin and incubated with the appropriate primary antibody (Ab). Gag was detected by a rabbit Ab recognizing CA (Seramun, Heidesee, Germany). The mouse anti-transferrin receptor antibody was purchased from Life Technologies and the anti-β-actin antibody from Sigma-Aldrich (St. Louis, MO, USA). The anti-mouse and anti-rabbit secondary antibodies coupled to HRP were obtained from Dianova (Hamburg, Germany). Tagged proteins were detected by monoclonal antibodies (mAb) directly conjugated to horse radish peroxidase (HRP) specific for HA (Roche, Basel, Switzerland) or FLAG (Sigma-Aldrich, St. Louis, MO, USA). Protein bands were quantified using AIDA [41 ].