Anti myc tag antibody

To analyze phosphorylation changes in Ncl by Phos‐tag SDS–PAGE, ectopically expressed myc‐tagged Ncl was first purified from iKras PDAC cells by immunoprecipitation. Briefly, empty vector control or Myc‐Ncl transfected iKras PDAC cells from 10 cm dishes were lysed in 1 ml of IP lysis buffer (50 mM Tris–HCl pH 7.4, 100 mM NaCl, 1% Igepal CA‐630, 0.1% SDS, 0.5% sodium deoxycholate, supplemented with phosphatase and protease inhibitor cocktails (Roche)), sonicated with a sonicator bath (Bioruptor Pico—Rm 343) for 15 cycles, and cleared by centrifugation (12,000 g, 10 min). The protein concentration of the cleared lysate was then measured and balanced, before immunoprecipitation with 4 μg of anti‐Myc‐tag antibody (Cell Signaling) per sample, preconjugated to protein G Dynabeads (Thermo). The beads were washed three times with 1 ml of lysis buffer, and the purified proteins were eluted by boiling the beads in 60 μl 2% SDS, 100 mM Tris/HCl pH 7.5, 0.1 M DTT for 5 min. The eluates were then subjected to Phos‐tag gel electrophoresis using Phos‐tag™ SuperSep™ 7.5% acrylamide precast gels, according to the manufacturer's instructions, before immunoblotting with anti‐Myc antibody to visualize the migration of Myc‐Ncl through the gels.

In preparation for immunoprecipitation, sepharose beads coated with the anti-myc-tag antibody (Cell Signaling Technology, MA, US) were blocked with 0.5% BSA in lysis buffer (150 mM NaCl, 50 mM Tris pH7.4, 1% Triton) for 1 hour at 4 °C. Then, cell lysate or growth medium containing myc-tagged BDNF was added to the beads and incubated at 4 °C for 2 hours, followed by 3 wash steps in lysis buffer and elution by boiling the beads in polyacrylamide gel loading buffer at 90 °C for 5 minutes.