Anti mcherry antibody 16d7

Cells were lysed in RIPA buffer containing protease inhibitors with glass beads using FastPrep (MP Biomedicals, CA) for 20s, twice, at speed 6. Cleared cell lysate was denatured with SDS buffer and separated by SDS–PAGE with Novex 4–12% Tris-glycine polyacrylamide gel (Invitrogen, Life Technologies, CA). Western blot analysis was performed using anti-mCherry antibody (16D7) at 1:2000 dilution (Thermo Fisher, MA), anti-GFP antibody (GF28R) at 1:2000 dilution (Thermo Fisher, MA) and anti–AtpB antibody at 1:5000 dilution (Agrisera, Sweden) as a loading control. HRP-conjugated secondary antibody was used to detect signal with SuperSignal West Femto (Thermo Fisher, MA). Image was acquired with Image Quant LAS 4000 (GE Healthcare, IL). The band intensity was quantified with ImageJ (NIH).

Transfected HEK293T cells (1.4x10⁶) or infected HFFs expressing mCherry/mCherryFEN1 (1x10⁶) were lysed for 20 min at 4°C in 800 μL of CoIP buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1 mM PMSF, 2 μg/mL of aprotinin, 2 μg/mL of leupeptin, and 2 μg/mL of pepstatin). After centrifugation, aliquots of each sample were taken as input controls and the remaining supernatant was incubated with anti-FLAG antibody M2 coupled to protein-A-sepharose beads or anti-mCherry antibody 16D7 (Thermo Fisher Scientific, Waltham, MA, USA) coupled to protein-G-dynabeads for 2 h at 4°C. The beads were collected by centrifugation and washed five times in 1 mL CoIP buffer. Finally, the immunoprecipitated proteins were recovered by boiling in 4xSDS sample buffer and protein complexes were analyzed by immunoblotting.