Pierce anti c myc agarose

HEK293T cells were transfected with a c-myc-tagged
human NCLX-encoding plasmid. Forty-eight hours posttransfection, cells were
incubated with forskolin or cotreated with H89 and forskolin and then lysed in
the presence of protease inhibitors (Sigma) and phosphatase inhibitors
(PhosSTOP; Roche), and the lysate was immunoprecipitated with
anti-c-myc agarose beads (Pierce Anti-c-Myc agarose; Thermo
Scientific) using the manufacturer’s protocol. On-bead tryptic
proteolysis and MS were performed as previously described (Fukuyama et al., 2012 (link)).

After the designed treatments, the cells were suspended with the immunoprecipitation (IP) lysis buffer (10 mM Tris-HCl, pH 7.4 [Vivantis Technologies, PB0852], 100 mM NaCl [Vivantis Technologies, PB0570], 2.5 mM MgCl₂, 0.5% Triton X-100 [Sigma-Aldrich, T8787], phosphatase inhibitor and proteinase inhibitor cocktail), followed by sonication and centrifuged at 12,000 g for 10 min. Part of the supernatant (1–1.5 mg) was transferred to a new tube and diluted with the IP lysis buffer to achieve the concentration at 1–1.5 μg/μl. The supernatant was incubated with either control IgG or antibodies following the instructions of antibody datasheets overnight with gentle rotation at 4°C and was subsequently incubated with 10 μl protein A/G (Thermo Fisher Scientific, 20,421) for additional 4 h. For FLAG IP or MYC IP, the supernatant was incubated with 10 μl anti-FLAG M2 Affinity Gel (Sigma-Aldrich, A2220) or Pierce™ Anti-c-Myc Agarose (Thermo Fisher Scientific, 20,168) and mixed overnight with gentle rotation at 4°C. Next, the immunoprecipitates were washed with IP lysis buffer 3 times. The immunoprecipitants were then eluted by boiling for 5 min in sample loading buffer (Bio-Rad Laboratories, 1,610,737) and analyzed with immunoblotting.