Pierce crosslink ip kit

For CoIP in HEK293s, cells were grown in six‐well plates to 50% confluency and cotransfected with Myc‐BMPR2 or Myc‐BMPR2‐S532X and HA‐β‐arrestin1/2 using Lipofectamine 2000 (Invitrogen). After 48 h of transfection, cells were scraped and lysed in lysis buffer of Pierce Crosslink IP Kit (Thermo/Pierce) with complete protease inhibitor cocktail tablet (Roche Applied Sciences). Cell lysates were incubated overnight at 4°C with anti‐Myc‐sepharose made with anti‐Myc antibody (Cell Signaling) and Pierce Crosslink IP Kit (Thermo/Pierce). Sepharose beads were then eluted with 0.5 mg/ml c‐Myc peptide (Genscript) in TBS. The elution samples were then detected by immunoblotting.

The Pierce^TM Crosslink IP kit (Thermo Fisher Scientific) was used to crosslink the BCL-XL antibody to 20 µL Protein A/G affinity beads. Beads were washed with 1X coupling buffer before incubation with end-over-end mixing at room temperature for 1 h with 3 µg BCL-XL antibody or rabbit IgG (#2729 s, Cell Signaling) dissolved in 1X coupling buffer. Bound antibodies were cross-linked to the Protein A/G beads using disuccinimidyl suberate by end-over-end mixing for 1 h at room temperature. Columns were centrifuged and washed with elution buffer and IP lysis/wash buffer, and stored at 4 °C. For co-immunoprecipitation, cells were treated as indicated in figure legends and lysed in RIPA lysis buffer. 500 µg of cell lysate was pre-cleared using control agarose beads for 1 h with end-over-end mixing, and the pre-cleared lysate was then incubated with the cross-linked antibody overnight at 4 °C. Bound protein complexes were eluted from the columns using elution buffer and precipitates were combined with 6X Laemlli buffer, boiled for 5 min at 95 °C and analysed via western blotting. Membranes were probed for BCL-XL, BIM, BAX, BAK and PUMA, with GAPDH as a loading control.