Mouse anti tnfr1

Western blot analysis was performed as described previously^{40 (link)} using the following antibodies: mouse anti-caspase-8, mouse anti-cFLIP (Alexis Biochemicals, Grünberg, Germany), mouse anti-FADD, mouse anti-XIAP (clone 28), mouse anti-RIP1 (BD Transduction Laboratories, Heidelberg, Germany), rabbit anti-Bid, rabbit anti-caspase-3, mouse anti-caspase-9 (Cell Signaling, Beverly, MA, USA), rabbit anti-TRAIL-R2 (Chemicon, Billerica, MA, USA), goat anti-cIAP1 (R&D Systems, Wiesbaden, Germany), rabbit anti-cIAP2 (Epitomics, Burlingname, CA, USA), mouse anti-TNFR1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Mouse anti-α-tubulin (Calbiochem, Darmstadt, Germany) or mouse anti-β-actin (Sigma) were used as loading controls. Goat anti-mouse IgG, donkey anti-goat IgG, goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology) and Goat anti-mouse IgG1 or Goat anti-mouse IgG2b (Southern Biotech, Birmingham, AL, USA) conjugated to horseradish peroxidase were used as secondary antibodies. Enhanced chemiluminescence was used for detection (Amersham Bioscience, Freiburg, Germany). Representative blots of at least two independent experiments are shown.

Primary antibodies used were rabbit anti‐p65 from Santa Cruz (clone C‐20, ref. sc‐372) at 1/250, mouse anti‐myc9E10 (developed by Bishop, J.M. was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the University of Iowa), mouse anti‐HA (Eurogentec, clone 16B12, ref. MMS‐101R), rabbit anti‐HA (Sigma, ref. H6908), rabbit anti‐GFP (Amsbio, ref. TP401), rabbit anti‐UBAP1 (Proteintech, ref. 12385‐1‐AP), mouse anti‐His (Sigma, clone HIS‐1, ref. H1029), mouse anti‐FLAG (Sigma, clone M2, ref. F1804) all at 1/1000 and mouse anti‐TNFR1 (Santa Cruz, clone H‐5, ref. sc‐8436) and rabbit anti‐TSG101 (Atlas Antibodies, ref. HPA006161) both at 1/200. Rabbit polyclonal anti‐PumA serum was obtained by repeated immunization of rabbits with purified PumA (Eurogentec) and was used at 1/1,000 for Western blot and for immunofluorescence microscopy. Purified BtpA was used to obtain chicken anti‐BtpA (Eurogentec). Anti‐EF‐Tu antibody (kind gift from R. Voulhoux) was used at 1/10,000.
Secondary antibodies used were anti‐rabbit, mouse, chicken or rat conjugated with Alexas‐488, ‐555 or ‐647 all from Jackson ImmunoResearch. When necessary, phalloidin‐568 (1/1,000) was used to label the actin cytoskeleton and DAPI nuclear dye (1/1,000) for the host cell nucleus. For Western blots, anti‐mouse or rabbit‐HRP antibodies were used at 1/5,000.